Preparation and application of fluorescent oligomer nanoparticle probes
By encapsulating oligomer molecules into nanoparticles and functionalizing them, the problems of photostability and water solubility of fluorescent probes were solved, and fluorescent oligomer nanoparticle probes with high brightness, high stability and uniform size were prepared, which are suitable for the fields of biomedicine and materials science.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2026-02-13
- Publication Date
- 2026-07-21
AI Technical Summary
Existing fluorescent probes suffer from poor photostability, easy bleaching and quenching, non-uniform size, and poor water solubility. In particular, the application of fluorescent oligomers in biological fluorescent probes lacks versatility.
The method of encapsulating oligomer molecules into nanoparticles involves preparing compound W-2 by reacting 2-bromofluorene with fluorene-2,7-diboronic acid pinacol ester, and then reacting it with a dibromo reagent to prepare small oligomer molecules. These small molecules are then mixed with functional polymers and silicon-oxygen precursors to prepare fluorescent oligomer nanoparticle probes, which are then functionalized by encapsulating them with a silica shell.
The prepared fluorescent oligomeric nanoparticle probes have high brightness, good stability, and uniform size, solving the problems of poor photostability and easy bleaching and quenching, while improving water solubility and functionalization effect.
Smart Images
Figure CN121699600B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of fluorescent oligomeric nanoparticle probes. Background Technology
[0002] With the advancement of hardware, the availability of fluorescent probes has become increasingly important. Fluorescent probes have become indispensable tools in molecular biology and medicine. Due to their high sensitivity and rapid response, fluorescent probes offer numerous benefits in visualizing spatiotemporal changes in biological systems. Traditional fluorescent probes include fluorescent small molecule probes and fluorescent nanoparticles. Fluorescent small molecule probes have advantages such as well-defined chemical structures and ease of functionalization, but suffer from poor photostability and susceptibility to bleaching and quenching. Fluorescent nanoparticles possess advantages such as high fluorescence brightness, excellent photostability, and a large surface area-to-volume ratio, allowing for the attachment of multiple reactive sites. However, they also have disadvantages such as size inhomogeneity and complex synthesis. Therefore, developing novel probes with higher brightness and photostability has significant research value and application potential in the fields of biomedicine and materials science.
[0003] Fluorescent oligomers are molecules composed of a few repeating fluorescent units covalently linked together. They have a well-defined structure and exhibit higher brightness and photostability compared to traditional small fluorescent molecules. However, oligomers, as bioluminescent probes, face challenges such as poor water solubility and a lack of binding sites with biomolecules. Solving these two problems is crucial for the preparation of fluorescent oligomer probes. Existing methods, based on the fluorene structure as the reaction site, introduce corresponding chemical groups through covalent bonding to achieve functionalization and water solubility of fluorescent oligomers, but these methods lack universality. Summary of the Invention
[0004] This invention employs a simple method to encapsulate oligomer molecules into nanoparticles of suitable size, thus solving the problems of water solubility and functionalization.
[0005] The purpose of this invention is to provide a fluorescent oligomer nanoparticle probe with high brightness, high stability, and more uniform size, and a method for preparing the same.
[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:
[0007] A method for preparing a fluorescent oligomeric nanoparticle probe, comprising:
[0008] Compound W-2 was prepared by reacting 2-bromofluorene with pinacol fluorene-2,7-diborate; the molar amount of pinacol fluorene-2,7-diborate used was 50-200% of the molar amount of 2-bromofluorene.
[0009] Oligomeric small molecules were prepared by reacting compound W-2 with a dibromine reagent;
[0010] Oligomeric small molecules are mixed with functional polymers and silicon oxide precursors to form nanoparticles. These nanoparticles are then treated with activated silica and functionalizing reagents to prepare fluorescent oligomeric nanoparticle probes. This invention synthesizes three different colored oligomeric small molecules, the structures of which are not disclosed. After further preparation into fluorescent oligomeric nanoparticle probes, these probes exhibit high brightness, high stability, and more uniform size, along with good sealing stability. They can also be used for confocal imaging of cells, providing a new solution to problems such as poor photostability and susceptibility to bleaching and quenching.
[0011] Preferably, the functional polymer includes PSMA, which is formulated as a PSMA solution for use, and the content of PSMA in the PSMA solution is 0.5-2 mg / mL.
[0012] Preferably, the silicon-oxygen precursor includes TMOS and / or TEOS;
[0013] TMOS is prepared as a TMOS solution for use, with a TMOS content of 0.5-2 mg / mL in the TMOS solution;
[0014] TEOS is prepared as a TEOS solution for use, with a TEOS content of 0.5-2 mg / mL.
[0015] Preferably, the activated silica is prepared by passing a sodium silicate solution through a hydrogen-form cation exchange resin.
[0016] Preferably, the functionalizing reagent includes sodium carboxyethylsilanetriol. When fluorescent oligomer molecules are prepared into nanoparticle probes, encapsulating them with a silica shell and functionalizing them can prevent aggregation-induced quenching of the fluorescent oligomer molecules. Furthermore, the doped silicon-oxygen precursor creates steric hindrance between molecules, effectively preventing excessive aggregation of fluorescent oligomer molecules and achieving higher fluorescence quantum yield. Fluorescent oligomer molecules may leak within the nanoparticles; encapsulating them with a silica shell effectively prevents fluorescence leakage. The surface of the nanoparticles is functionalized with sodium carboxyethylsilanetriol to create coupling sites, followed by modification with streptavidin and antibodies, improving coupling effectiveness while preventing fluorescence leakage.
[0017] Preferably, the functionalizing agent includes 3-aminopropyltrihydroxysilane. The present invention can also use 3-aminopropyltrihydroxysilane and sodium carboxyethylsilanetriol as functional agents, which can improve the dispersibility and long-term stability of fluorescent oligomeric nanoparticle probes and reduce leakage of small oligomeric molecules.
[0018] Preferably, the dibromo reagent includes 2,7-dibromofluorene, and the oligomer is a blue oligomer B-1.
[0019] Preferably, the dibromo reagent includes 4,7-dibromo-2,1,3-benzothiadiazole, and the oligomer is a yellow oligomer Y-1.
[0020] Preferably, the dibromo reagent includes 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole, and the oligomer is a yellow oligomer R-1.
[0021] This invention discloses fluorescent oligomeric nanoparticle probes prepared by the above method.
[0022] Preferably, in the preparation of compound W-2, 2-bromofluorene is mixed with 1,4-dioxane, and then fluorene-2,7-diborate pinacol ester, tetra-triphenylphosphine palladium and sodium carbonate aqueous solution are added. Under a nitrogen atmosphere, the mixture is stirred at 70-90°C for 6-24 h. After the reaction is complete, the solvent is removed by vacuum distillation, and then purified by eluent to obtain compound W-2.
[0023] More preferably, in the preparation of compound W-2, the molar volume ratio of 2-bromofluorene to 1,4-dioxane is 1-10 mmol: 1-10 mL.
[0024] More preferably, in the preparation of compound W-2, the molar amount of fluorene-2,7-diboronic acid pinacol ester used is 50-200% of the molar amount of 2-bromofluorene used.
[0025] More preferably, in the preparation of compound W-2, the molar amount of tetratriphenylphosphine palladium used is 2-10% of the molar amount of 2-bromofluorene used.
[0026] More preferably, in the preparation of compound W-2, the sodium carbonate aqueous solution is composed of a mixture of sodium carbonate and water, the sodium carbonate content in the sodium carbonate aqueous solution is 1-5 mol / L, and the molar volume ratio of 2-bromofluorene to the sodium carbonate aqueous solution is 1-10 mmol: 1-4 mL.
[0027] More preferably, in the preparation of compound W-2, the eluent is a mixture of PE and EA, wherein PE and EA are mixed in a volume ratio of 10-30:0.5-2.
[0028] Preferably, in the preparation of the blue oligomer small molecule, 2,7-dibromofluorene is mixed with 1,4-dioxane, and then W-2, tetraphenylphosphine palladium and sodium carbonate aqueous solution are added. Under a nitrogen atmosphere, the mixture is stirred at 70-90℃ for 6-24h. After the reaction is completed, the solvent is removed by vacuum distillation, and then purified by eluent to obtain blue oligomer B-1, i.e., blue oligomer small molecule.
[0029] More preferably, in the preparation of the blue oligomer small molecules, the molar volume ratio of 2,7-dibromofluorene to 1,4-dioxane is 1-10 mmol: 1-10 mL.
[0030] More preferably, in the preparation of the blue oligomer small molecules, the molar amount of W-2 used is 100-300% of the molar amount of 2,7-dibromofluorene used.
[0031] More preferably, in the preparation of the blue oligomer small molecules, the molar amount of tetratriphenylphosphine palladium used is 2-10% of the molar amount of 2,7-dibromofluorene used.
[0032] More preferably, in the preparation of the blue oligomer small molecules, the sodium carbonate aqueous solution is composed of a mixture of sodium carbonate and water, and the sodium carbonate content in the sodium carbonate aqueous solution is 1-5 mol / L.
[0033] More preferably, in the preparation of the blue oligomer small molecules, the molar volume ratio of 2,7-dibromofluorene to sodium carbonate aqueous solution is 1-10 mmol: 1-4 mL.
[0034] More preferably, in the preparation of the blue oligomer small molecules, the eluent is a mixture of PE and EA, wherein PE and EA are mixed in a volume ratio of 10-30:0.5-2.
[0035] Preferably, in the preparation of the yellow oligomer small molecule, 4,7-dibromo-2,1,3-benzothiadiazole is mixed with 1,4-dioxane, and then W-2, tetraphenylphosphine palladium and sodium carbonate aqueous solution are added. Under a nitrogen atmosphere, the mixture is stirred at 70-90℃ for 6-24h. After the reaction is completed, the solvent is removed by vacuum distillation, and then purified by eluent to obtain the yellow oligomer small molecule Y-1, i.e., the yellow oligomer small molecule.
[0036] More preferably, in the preparation of the yellow oligomer small molecule, the molar volume ratio of 4,7-dibromo-2,1,3-benzothiadiazole to 1,4-dioxane is 1-10 mmol: 1-10 mL.
[0037] More preferably, in the preparation of the yellow oligomer small molecule, the molar amount of W-2 used is 100-300% of the molar amount of 4,7-dibromo-2,1,3-benzothiadiazole used.
[0038] More preferably, in the preparation of the yellow oligomer small molecule, the molar amount of tetratriphenylphosphine palladium used is 2-10% of the molar amount of 4,7-dibromo-2,1,3-benzothiadiazole used.
[0039] More preferably, in the preparation of the yellow oligomer small molecules, the sodium carbonate aqueous solution is composed of a mixture of sodium carbonate and water, and the sodium carbonate content in the sodium carbonate aqueous solution is 1-5 mol / L.
[0040] More preferably, in the preparation of the yellow oligomer small molecule, the molar volume ratio of 4,7-dibromo-2,1,3-benzothiadiazole to sodium carbonate aqueous solution is 1-10 mmol: 1-4 mL.
[0041] More preferably, in the preparation of the yellow oligomer small molecules, the eluent is a mixture of PE and EA, wherein PE and EA are mixed in a volume ratio of 10-30:0.5-2.
[0042] Preferably, in the preparation of the red oligomer small molecule, 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole is mixed with 1,4-dioxane, and then W-2, tetra-triphenylphosphine palladium and sodium carbonate aqueous solution are added. Under a nitrogen atmosphere, the mixture is stirred at 70-90℃ for 6-24h. After the reaction is completed, the solvent is removed by vacuum distillation, and then purified by eluent to obtain red oligomer R-1, i.e., red oligomer small molecule.
[0043] More preferably, in the preparation of the red oligomer small molecule, the molar volume ratio of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole to 1,4-dioxane is 1-10 mmol: 1-10 mL.
[0044] More preferably, in the preparation of the red oligomer small molecule, the molar amount of W-2 used is 100-300% of the molar amount of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole used.
[0045] More preferably, in the preparation of the red oligomer small molecule, the molar amount of tetratriphenylphosphine palladium used is 2-10% of the molar amount of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole used.
[0046] More preferably, in the preparation of the red oligomer small molecules, the sodium carbonate aqueous solution is composed of a mixture of sodium carbonate and water, and the sodium carbonate content in the sodium carbonate aqueous solution is 1-5 mol / L.
[0047] More preferably, in the preparation of the red oligomer small molecule, the molar volume ratio of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole to sodium carbonate aqueous solution is 1-10 mmol: 1-4 mL.
[0048] More preferably, in the preparation of the red oligomer small molecules, the eluent is a mixture of PE and EA, wherein PE and EA are mixed in a volume ratio of 10-30:0.5-2.
[0049] Preferably, in the preparation of the blue fluorescent oligomeric nanoparticle probe, a small blue oligomeric molecule is added to tetrahydrofuran and mixed to obtain a small blue oligomeric molecule solution; then, the functional polymer PSMA is added to tetrahydrofuran and mixed to obtain a PSMA solution; the silicon-oxygen precursor TMOS is added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS is added to tetrahydrofuran and mixed to obtain a TEOS solution; the small blue oligomeric molecule solution, PSMA solution, TMOS solution, and TEOS solution are added to tetrahydrofuran and mixed to obtain a mixed solution; an ammonia solution is added to an ultrasonic water bath and ultrasonicated for 1-5 min; then, the solution is concentrated by blowing nitrogen gas at 80-100℃; the solution is filtered through a 220 nm filter while hot; then, activated silica is added and stirred for 6-24 h; then, a functionalizing reagent is added and stirred for 2-8 h for functionalization treatment to prepare the blue fluorescent oligomeric nanoparticle probe.
[0050] More preferably, in the preparation of the blue fluorescent oligomer nanoparticle probe, the content of the blue oligomer small molecule in the blue oligomer small molecule solution is 0.5-2 mg / mL.
[0051] More preferably, in the preparation of the blue fluorescent oligomeric nanoparticle probe, the content of PSMA in the PSMA solution is 0.5-2 mg / mL, the content of TMOS in the TMOS solution is 0.5-2 mg / mL, and the content of TEOS in the TEOS solution is 0.5-2 mg / mL.
[0052] More preferably, in the preparation of the blue fluorescent oligomer nanoparticle probe, the volume of the blue oligomer small molecule solution used in the mixed solution is 10-20% of the volume of tetrahydrofuran used.
[0053] More preferably, in the preparation of the blue fluorescent oligomeric nanoparticle probe, the volume of PSMA solution used in the mixed solution is 50-200% of the volume of blue oligomeric small molecule solution, the volume of TMOS solution used in the mixed solution is 50-200% of the volume of blue oligomeric small molecule solution, and the volume of TEOS solution used in the mixed solution is 50-200% of the volume of blue oligomeric small molecule solution.
[0054] More preferably, in the preparation of the blue fluorescent oligomer nanoparticle probe, the ammonia solution is prepared by adding ammonia to water to adjust the pH to 10-11, and the volume of the ammonia solution used is 400-600% of the volume of the mixed solution.
[0055] More preferably, in the preparation of the blue fluorescent oligomer nanoparticle probe, nitrogen gas is blown and concentrated to the volume of the mixed solution, and the activated silica is a solution prepared from sodium silicate solution through a hydrogen-type cation exchange resin, wherein the sodium silicate content in the sodium silicate solution is 0.1-1 wt%, and the volume of activated silica used is 200-300 times that of the mixed solution.
[0056] More preferably, in the preparation of the blue fluorescent oligomeric nanoparticle probe, the functionalizing agent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.1-1 wt% of the mixed solution.
[0057] Preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the yellow oligomer small molecules are added to tetrahydrofuran and mixed to obtain a yellow oligomer small molecule solution; then the functional polymer PSMA is added to tetrahydrofuran and mixed to obtain a PSMA solution; the silicon-oxygen precursor TMOS is added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS is added to tetrahydrofuran and mixed to obtain a TEOS solution; the yellow oligomer small molecule solution, PSMA solution, TMOS solution and TEOS solution are added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution is added to an ultrasonic water bath and ultrasonicated for 1-5 min; then the solution is concentrated by blowing nitrogen gas at 80-100℃; the solution is filtered through a 220 nm filter while hot; then activated silica is added and stirred for 6-24 h; then functionalizing reagent is added and stirred for 2-8 h for functionalization treatment to prepare the yellow fluorescent oligomer nanoparticle probe.
[0058] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the content of the yellow oligomer small molecule in the yellow oligomer small molecule solution is 0.5-2 mg / mL; the content of PSMA in the PSMA solution is 0.5-2 mg / mL; the content of TMOS in the TMOS solution is 0.5-2 mg / mL; and the content of TEOS in the TEOS solution is 0.5-2 mg / mL.
[0059] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the volume of the yellow oligomer small molecule solution used in the mixed solution is 10-20% of the volume of tetrahydrofuran used.
[0060] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the volume of PSMA solution used in the mixed solution is 50-200% of the volume of the yellow oligomer small molecule solution used.
[0061] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the volume of TMOS solution used in the mixed solution is 50-200% of the volume of the yellow oligomer small molecule solution used, and the volume of TEOS solution used in the mixed solution is 50-200% of the volume of the yellow oligomer small molecule solution used.
[0062] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the ammonia solution is prepared by adding ammonia to water to adjust the pH to 10-11, and the volume of the ammonia solution used is 400-600% of the volume of the mixed solution.
[0063] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, nitrogen gas is blown and concentrated to the volume of the mixed solution, and the activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-type cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.1-1 wt%, and the volume of activated silica used is 200-300 times that of the mixed solution.
[0064] More preferably, in the preparation of the yellow fluorescent oligomer nanoparticle probe, the functionalizing agent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.1-1 wt% of the mixed solution.
[0065] Preferably, in the preparation of the red fluorescent oligomeric nanoparticle probe, red oligomeric small molecules are added to tetrahydrofuran and mixed to obtain a red oligomeric small molecule solution; then, functional polymer PSMA is added to tetrahydrofuran and mixed to obtain a PSMA solution; silicon-oxygen precursor TMOS is added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS is added to tetrahydrofuran and mixed to obtain a TEOS solution; the red oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution are added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution is added to an ultrasonic water bath and ultrasonicated for 1-5 min; then the solution is concentrated by blowing nitrogen gas at 80-100℃; the solution is filtered through a 220 nm filter while hot; then activated silica is added and stirred for 6-24 h; then functionalizing reagent is added and stirred for 2-8 h for functionalization treatment to prepare the fluorescent oligomeric nanoparticle probe.
[0066] More preferably, in the preparation of the red fluorescent oligomer nanoparticle probe, the content of red oligomer small molecules in the red oligomer small molecule solution is 0.5-2 mg / mL, the content of PSMA in the PSMA solution is 0.5-2 mg / mL, the content of TMOS in the TMOS solution is 0.5-2 mg / mL, and the content of TEOS in the TEOS solution is 0.5-2 mg / mL.
[0067] More preferably, in the preparation of the red fluorescent oligomer nanoparticle probe, the volume of the red oligomer small molecule solution used in the mixed solution is 10-20% of the volume of tetrahydrofuran used.
[0068] More preferably, in the preparation of the red fluorescent oligomer nanoparticle probe, the volume of PSMA solution used in the mixed solution is 50-200% of the volume of red oligomer small molecule solution used, the volume of TMOS solution used in the mixed solution is 50-200% of the volume of red oligomer small molecule solution used, and the volume of TEOS solution used in the mixed solution is 50-200% of the volume of red oligomer small molecule solution used.
[0069] More preferably, in the preparation of the red fluorescent oligomer nanoparticle probe, the ammonia solution is prepared by adding ammonia to water to adjust the pH to 10-11, and the volume of the ammonia solution used is 400-600% of the volume of the mixed solution.
[0070] More preferably, in the preparation of the red fluorescent oligomer nanoparticle probe, nitrogen gas is blown and concentrated to the volume of the mixed solution, and the activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-type cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.1-1 wt%, and the volume of activated silica used is 200-300 times that of the mixed solution.
[0071] More preferably, in the preparation of the red fluorescent oligomeric nanoparticle probe, the functionalizing agent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.1-1 wt% of the mixed solution.
[0072] More preferably, the amount of sodium carboxyethylsilanetriol salt used is 0.2-1 wt% of the mixed solution.
[0073] More preferably, the amount of 3-aminopropyltrihydroxysilane used is 0.1-0.6 wt% of the mixed solution.
[0074] More preferably, the amount of 1,1,3,3-tetramethyl-1,3-disiloxanediol used is 0.05-0.3 wt% of the mixed solution. In addition to sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane, this invention can also use 1,1,3,3-tetramethyl-1,3-disiloxanediol as a functional agent. In the presence of sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane, the use of 1,1,3,3-tetramethyl-1,3-disiloxanediol can improve the dispersibility and long-term stability of the fluorescent oligomer nanoparticle probe, and reduce the leakage of small oligomer molecules.
[0075] This invention utilizes a method to prepare compound W-2 by reacting 2-bromofluorene and fluorene-2,7-diboronic acid pinacol ester. Then, it reacts with different compounds to prepare oligomers of different colors. For example, compound W-2 reacts with 2,7-dibromofluorene to prepare a blue oligomer, with 4,7-dibromo-2,1,3-benzothiadiazole to prepare a yellow oligomer, and with 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole to prepare a red oligomer. These different colored oligomers are then mixed with a silicon-oxygen precursor and a functional polymer to prepare nanoparticles via a nanoprecipitation method. Finally, fluorescent oligomer nanoparticle probes are prepared by encapsulating the nanoparticles with a silica shell and performing functionalization. Therefore, these probes exhibit the following advantages: high brightness, high stability, uniform size, and good sealing effect on the oligomers. Therefore, the present invention provides a fluorescent oligomer nanoparticle probe with high brightness, high stability, and more uniform size, and a method for preparing the same. Attached Figure Description
[0076] Figure 1 This is the hydrogen NMR spectrum of W-2.
[0077] Figure 2 The image shows the hydrogen NMR spectrum of the blue oligomer molecule.
[0078] Figure 3 The image shows the hydrogen NMR spectrum of the yellow oligomer molecule.
[0079] Figure 4 This is the hydrogen NMR spectrum of the red oligomer molecule.
[0080] Figure 5 The images show the particle size and potential spectra of three fluorescent oligomeric nanoparticles.
[0081] Figure 6 These are TEM images of three fluorescent oligomeric nanoparticles.
[0082] Figure 7 The absorption and emission spectra of three fluorescent oligomeric nanoparticles are shown.
[0083] Figure 8 This is a fluorescence intensity diagram.
[0084] Figure 9 This image shows three fluorescent oligomeric nanoparticles labeled with cellular microtubules. Detailed Implementation
[0085] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0086] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0087] Example 1: A method for preparing compound W-2
[0088] Preparation of compound W-2: 2-bromofluorene was mixed with 1,4-dioxane, then fluorene-2,7-diboron pinacol ester, tetraphenylphosphine palladium, and sodium carbonate aqueous solution were added. The mixture was stirred at 80°C for 12 h under a nitrogen atmosphere. After the reaction was complete, the solvent was removed by vacuum distillation, and then purified using an eluent to obtain compound W-2. The molar amount of 2-bromofluorene used was 1 mmol, and the molar volume ratio of 2-bromofluorene to 1,4-dioxane was 1 mmol: 10 mL. The molar amount of pinacol diborate used is 100% of the molar amount of 2-bromofluorene used, the molar amount of tetra-triphenylphosphine palladium used is 5% of the molar amount of 2-bromofluorene used, the sodium carbonate aqueous solution is a mixture of sodium carbonate and water, the sodium carbonate content in the sodium carbonate aqueous solution is 2 mol / L, the molar volume ratio of 2-bromofluorene to sodium carbonate aqueous solution is 1 mmol: 2 mL, the eluent is a mixture of PE and EA, the PE and EA in the eluent are mixed in a volume ratio of 20:1.
[0089] The structure of compound W-2 is as follows:
[0090] .
[0091] The 1H NMR spectrum of compound W-2 is shown below. Figure 1 As shown, where, 1 H NMR (400 MHz, CDCl3) δ 7.93–7.28 (m, 1H), 3.98 (s, 2H), 1.38 (s, 5H).
[0092] Example 2: A method for preparing a small blue oligomer molecule
[0093] Preparation of blue oligomer small molecules: 2,7-dibromofluorene and 1,4-dioxane were mixed, and then W-2, tetra-triphenylphosphine palladium and sodium carbonate aqueous solution were added. The mixture was stirred at 80°C for 12 h under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by vacuum distillation, and then purified by eluent to obtain blue oligomer B-1, i.e., blue oligomer small molecules. The molar amount of 2,7-dibromofluorene used was 1 mmol, and the molar volume ratio of 2,7-dibromofluorene to 1,4-dioxane was 1 mmol: 10 mL. The molar amount of W-2 was 200% of the molar amount of 2,7-dibromofluorene used. The molar amount of tetra-triphenylphosphine palladium used was 5% of the molar amount of 2,7-dibromofluorene used. The sodium carbonate aqueous solution was prepared by mixing sodium carbonate and water, with a sodium carbonate content of 2 mol / L. The molar volume ratio of 2,7-dibromofluorene to the sodium carbonate aqueous solution was 1 mmol: 2 mL. The eluent was prepared by mixing PE and EA in a volume ratio of 10:1. W-2 is from Example 1.
[0094] The structure of blue oligomer B-1 is as follows:
[0095] .
[0096] The 1H NMR spectrum of the blue oligomer small molecules is as follows: Figure 2 As shown, where, 1 H NMR (400 MHz, CDCl3) δ7.65–7.27 (m, 1H), 3.95 (s, 0H), 2.47–2.38 (m, 0H), 2.02 (dd, J=10.1, 6.5 Hz, 0H), 1.02 (d, J=4.1 Hz, 2H), 0.71 (d, J=7.1 Hz, 1H).
[0097] Example 3: A method for preparing a small yellow oligomer
[0098] Preparation of yellow oligomer small molecules: 4,7-dibromo-2,1,3-benzothiadiazole was mixed with 1,4-dioxane, and then W-2, tetraphenylphosphine palladium and sodium carbonate aqueous solution were added. The mixture was stirred at 80°C for 12 h under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by vacuum distillation, and then purified by eluent to obtain yellow oligomer small molecule Y-1, i.e., yellow oligomer small molecule. The molar amount of 4,7-dibromo-2,1,3-benzothiadiazole used was 1 mmol, and the molar volume ratio of 4,7-dibromo-2,1,3-benzothiadiazole to 1,4-dioxane was 1 mmol: 10 mL. The molar amount of W-2 used was 200% of the molar amount of 4,7-dibromo-2,1,3-benzothiadiazole used. The molar amount of tetraphenylphosphine palladium used was 5% of the molar amount of 4,7-dibromo-2,1,3-benzothiadiazole used. The sodium carbonate aqueous solution was prepared by mixing sodium carbonate and water, and the sodium carbonate content in the sodium carbonate aqueous solution was 2 mol / L. The molar volume ratio of 4,7-dibromo-2,1,3-benzothiadiazole to the sodium carbonate aqueous solution was 1 mmol: 2 mL. The eluent was prepared by mixing PE and EA in a volume ratio of 10:1. W-2 was derived from Example 1.
[0099] The structure of the yellow oligomer Y-1 is as follows:
[0100] .
[0101] The 1H NMR spectrum of the yellow oligomer molecules is as follows: Figure 3 As shown, where, 1 H NMR (400 MHz, CDCl3) δ 7.97–7.23 (m, 1H), 3.96 (s, 1H), 2.43 (tq, J=13.6, 6.9 Hz, 2H), 1.72–1.53 (m, 2H).
[0102] Example 4: A method for preparing a red oligomer small molecule
[0103] Preparation of the red oligomer: 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole was mixed with 1,4-dioxane, and then W-2, tetra(triphenylphosphine)palladium, and an aqueous solution of sodium carbonate were added. The mixture was stirred at 80°C for 12 h under a nitrogen atmosphere. After the reaction was complete, the solvent was removed by vacuum distillation, and then purified using an eluent to obtain the red oligomer R-1, i.e., the red oligomer molecule. The molar amount of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole used was 1 mmol, and the molar volume ratio of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole to 1,4-dioxane used was 1 mmol. 10 mL of W-2 was used, representing 200% of the molar amount of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole. The molar amount of tetra-triphenylphosphine palladium was 5% of the molar amount of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole. The sodium carbonate aqueous solution was prepared by mixing sodium carbonate and water, with a sodium carbonate content of 2 mol / L. The molar volume ratio of 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole to the sodium carbonate aqueous solution was 1 mmol: 2 mL. The eluent was prepared by mixing PE and EA in a volume ratio of 10:1. W-2 was derived from Example 1.
[0104] The structure of red oligomer R-1 is as follows:
[0105] .
[0106] The 1H NMR spectrum of the red oligomer small molecules is as follows: Figure 4 As shown, where, 1 H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 4.02 (s, 2H), 2.86–2.77 (m, 2H), 2.46 (dh, J=13.6, 6.4 Hz, 5H), 1.56 (s, 12H), 1.32 (s, 21H).
[0107] Example 5: A method for preparing a blue fluorescent oligomer nanoparticle probe
[0108] Preparation of blue fluorescent oligomeric nanoparticle probes: Blue oligomeric small molecules were added to tetrahydrofuran and mixed to obtain a blue oligomeric small molecule solution; then, functional polymer PSMA was added to tetrahydrofuran and mixed to obtain a PSMA solution; silicon-oxygen precursor TMOS was added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS was added to tetrahydrofuran and mixed to obtain a TEOS solution; the blue oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution were added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution was added to an ultrasonic water bath and ultrasonicated for 2 min; then the solution was concentrated by blowing nitrogen at 90℃; the solution was filtered through a 220 nm filter while hot; then activated silica was added and stirred for 12 h; then functionalizing reagent was added and stirred for 4 h for functionalization treatment to obtain blue fluorescent oligomeric nanoparticle probes. The volume of the blue oligomer small molecule solution used was 200 μL, and the concentration of the blue oligomer small molecule in the solution was 1 mg / mL. The PSMA solution contained 1 mg / mL of PSMA, the TMOS solution contained 1 mg / mL of TMOS, and the TEOS solution contained 1 mg / mL of TEOS. The volume of the blue oligomer small molecule solution used in the mixed solution was 16.67% of the volume of tetrahydrofuran used. The volume of the PSMA solution used in the mixed solution was 100% of the volume of the blue oligomer small molecule solution used. The volume of the TMOS solution used in the mixed solution was [missing information - likely a percentage of the blue oligomer small molecule solution volume]. The volume of TEOS solution used in the mixed solution is 100% of the volume of the blue oligomer small molecule solution. The ammonia solution is prepared by adding ammonia to water to adjust the pH to 11, and the volume of the ammonia solution used is 500% of the volume of the mixed solution. Nitrogen gas is blown to concentrate to the volume of the mixed solution. The activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-form cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.54 wt%, and the volume of activated silica used is 250 times that of the mixed solution. The functionalizing reagent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution.
[0109] Example 6: A method for preparing a yellow fluorescent oligomer nanoparticle probe
[0110] Preparation of yellow fluorescent oligomeric nanoparticle probes: Yellow oligomeric small molecules were added to tetrahydrofuran and mixed to obtain a yellow oligomeric small molecule solution; then, functional polymer PSMA was added to tetrahydrofuran and mixed to obtain a PSMA solution; silicon-oxygen precursor TMOS was added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS was added to tetrahydrofuran and mixed to obtain a TEOS solution; the yellow oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution were added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution was added to an ultrasonic water bath and ultrasonicated for 2 min; then the solution was concentrated by nitrogen blowing at 90℃; the solution was filtered through a 220 nm filter while hot; then activated silica was added and stirred for 12 h; then functionalizing reagent was added and stirred for 4 h for functionalization treatment to obtain yellow fluorescent oligomeric nanoparticle probes. The volume of the yellow oligomer small molecule solution used was 200 μL, and the concentration of the yellow oligomer small molecule in the solution was 1 mg / mL. The PSMA solution contained 1 mg / mL of PSMA, the TMOS solution contained 1 mg / mL of TMOS, and the TEOS solution contained 1 mg / mL of TEOS. The volume of the yellow oligomer small molecule solution used in the mixed solution was 16.67% of the volume of tetrahydrofuran used. The volume of the PSMA solution used in the mixed solution was 100% of the volume of the yellow oligomer small molecule solution used. The volume of the TMOS solution used in the mixed solution was [missing information - likely a percentage of the volume of the yellow oligomer small molecule solution]. The volume of TEOS solution used in the mixed solution is 100% of the volume of the yellow oligomer small molecule solution. The ammonia solution is prepared by adding ammonia to water to adjust the pH to 11, and the volume of the ammonia solution used is 500% of the volume of the mixed solution. Nitrogen gas is blown to concentrate to the volume of the mixed solution. The activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-form cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.54 wt%, and the volume of activated silica used is 250 times that of the mixed solution. The functionalizing reagent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution.
[0111] Example 7: A method for preparing a red fluorescent oligomer nanoparticle probe
[0112] Preparation of red fluorescent oligomeric nanoparticle probes: Red oligomeric small molecules were added to tetrahydrofuran and mixed to obtain a red oligomeric small molecule solution; then, functional polymer PSMA was added to tetrahydrofuran and mixed to obtain a PSMA solution; silicon-oxygen precursor TMOS was added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS was added to tetrahydrofuran and mixed to obtain a TEOS solution; the red oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution were added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution was added to an ultrasonic water bath and ultrasonicated for 2 min; then the solution was concentrated by nitrogen blowing at 90℃; the solution was filtered through a 220 nm filter while hot; then activated silica was added and stirred for 12 h; then functionalizing reagent was added and stirred for 4 h for functionalization treatment to obtain red fluorescent oligomeric nanoparticle probes. The volume of the red oligomer small molecule solution used was 200 μL, with a concentration of 1 mg / mL. The PSMA solution contained 1 mg / mL of PSMA, the TMOS solution contained 1 mg / mL of TMOS, and the TEOS solution contained 1 mg / mL of TEOS. The volume of the red oligomer small molecule solution in the mixed solution was 16.67% of the volume of tetrahydrofuran. The volume of the PSMA solution in the mixed solution was 100% of the volume of the red oligomer small molecule solution. The volume of the TMOS solution in the mixed solution was [missing information - likely a percentage]. The volume of TEOS solution used in the mixed solution is 100% of the volume of the red oligomer small molecule solution. The ammonia solution is prepared by adding ammonia to water to adjust the pH to 11, and the volume of the ammonia solution used is 500% of the volume of the mixed solution. Nitrogen gas is blown to concentrate to the volume of the mixed solution. The activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-form cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.54 wt%, and the volume of activated silica used is 250 times that of the mixed solution. The functionalizing reagent is sodium carboxyethylsilanetriol, and the amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution.
[0113] Example 8: A method for preparing a blue fluorescent oligomer nanoparticle probe
[0114] Preparation of blue fluorescent oligomeric nanoparticle probes: Blue oligomeric small molecules were added to tetrahydrofuran and mixed to obtain a blue oligomeric small molecule solution; then, functional polymer PSMA was added to tetrahydrofuran and mixed to obtain a PSMA solution; silicon-oxygen precursor TMOS was added to tetrahydrofuran and mixed to obtain a TMOS solution; TEOS was added to tetrahydrofuran and mixed to obtain a TEOS solution; the blue oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution were added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution was added to an ultrasonic water bath and ultrasonicated for 2 min; then the solution was concentrated by blowing nitrogen at 90℃; the solution was filtered through a 220 nm filter while hot; then activated silica was added and stirred for 12 h; then functionalizing reagent was added and stirred for 4 h for functionalization treatment to obtain blue fluorescent oligomeric nanoparticle probes. The volume of the blue oligomer small molecule solution used was 200 μL, and the concentration of blue oligomer small molecules in the solution was 1 mg / mL; the PSMA solution contained 1 mg / mL PSMA, the TMOS solution contained 1 mg / mL TMOS, and the TEOS solution contained 1 mg / mL TEOS. The volume of the blue oligomer small molecule solution in the mixed solution was 16.67% of the volume of tetrahydrofuran, the volume of the PSMA solution in the mixed solution was 100% of the volume of the blue oligomer small molecule solution, the volume of the TMOS solution in the mixed solution was 100% of the volume of the blue oligomer small molecule solution, and the volume of the TEOS solution in the mixed solution was... The volume of the solution used is 100% of the volume of the blue oligomer small molecule solution. The ammonia solution is prepared by adding ammonia to water to adjust the pH to 11, and the volume of the ammonia solution used is 500% of the volume of the mixed solution. Nitrogen gas is blown to concentrate to the volume of the mixed solution. The activated silica is a solution prepared by passing sodium silicate solution through a hydrogen-form cation exchange resin. The sodium silicate content in the sodium silicate solution is 0.54 wt%, and the volume of the activated silica used is 250 times that of the mixed solution. The functionalizing reagents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.5 wt% of the mixed solution.
[0115] Example 9: A method for preparing a blue fluorescent oligomer nanoparticle probe
[0116] The difference between this embodiment and Example 8 lies in the use of functionalizing reagents. The functionalizing reagents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.2 wt% of the mixed solution.
[0117] Example 10: A method for preparing a blue fluorescent oligomer nanoparticle probe
[0118] The difference between this embodiment and Example 8 lies in the use of functionalizing reagents. The functionalizing reagents are sodium carboxyethylsilanetriol, 1,1,3,3-tetramethyl-1,3-disiloxanediol, and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution, the amount of 1,1,3,3-tetramethyl-1,3-disiloxanediol used is 0.5 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.2 wt% of the mixed solution.
[0119] Example 11: A method for preparing a blue fluorescent oligomer nanoparticle probe
[0120] The difference between this embodiment and Example 8 lies in the use of functionalizing reagents. The functionalizing reagents are sodium carboxyethylsilanetriol, 1,1,3,3-tetramethyl-1,3-disiloxanediol, and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution, the amount of 1,1,3,3-tetramethyl-1,3-disiloxanediol used is 0.5 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.08 wt% of the mixed solution.
[0121] Comparative Example 1:
[0122] The difference between this comparative example and Example 8 lies in the use of functionalizing agents. The functionalizing agents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.5 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.08 wt% of the mixed solution.
[0123] Comparative Example 2:
[0124] The difference between this comparative example and Example 8 lies in the use of functionalizing agents. The functionalizing agents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.05 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.5 wt% of the mixed solution.
[0125] Comparative Example 3:
[0126] The difference between this comparative example and Example 8 lies in the use of functionalizing agents. The functionalizing agents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane. The amount of sodium carboxyethylsilanetriol used is 0.05 wt% of the mixed solution, and the amount of 3-aminopropyltrihydroxysilane used is 0.08 wt% of the mixed solution.
[0127] Experimental example:
[0128] The fluorescent oligomeric nanoparticle probes prepared in Examples 5-7 were characterized by DLS in this invention, and the results are as follows: Figure 5 As shown, a is the potential spectrum, b is the particle size spectrum, and the fluorescent oligomeric nanoparticle probes include blue fluorescent oligomeric nanoparticle probes, yellow fluorescent oligomeric nanoparticle probes, and red fluorescent oligomeric nanoparticle probes. Figure 5 As shown in Figure a, the potentials of the three fluorescent oligomer nanoparticle probes are all below -20mV, proving that a large number of negatively charged groups are successfully attached to the surface of the three fluorescent oligomer nanoparticle probes, and they have good stability. Figure 5 As shown in Figure b, the particle sizes of the three fluorescent oligomeric nanoparticle probes are basically distributed in the range of 20-30 nm, which is suitable for immunofluorescence labeling.
[0129] The fluorescent oligomeric nanoparticle probes prepared in Examples 5-7 were characterized by TEM in this invention, and the results are as follows: Figure 6 As shown, a is a blue fluorescent oligomeric nanoparticle probe, b is a yellow fluorescent oligomeric nanoparticle probe, and c is a red fluorescent oligomeric nanoparticle probe. All three fluorescent oligomeric nanoparticle probes are regular spherical and their sizes are consistent with the DLS results.
[0130] The fluorescent oligomeric nanoparticle probes prepared in Examples 5-7 were characterized for their resistance to fluorescence leakage, and the results are as follows: Figure 7 As shown, a is the absorption spectrum and b is the emission spectrum. From a, it can be seen that the absorption range of the three nanoparticles basically matches the excitation wavelength of lasers commonly used in microscopes. Figure 9 -b indicates that the emission wavelengths of the three nanoparticles show that they can emit blue, yellow, and red light in the visible light range, achieving the design goal.
[0131] One mL of each of the three colors of fluorescent oligomeric nanoparticle probe aqueous solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 1000 g for 3 minutes. The lower layer filtrate was collected. Then, under the same conditions, the fluorescence intensity of the lower layer filtrate and the oligomeric small molecule nanoparticle aqueous solutions prepared in Examples 2-4 were tested to determine the leakage of the fluorescent oligomeric material. The results, as shown in the figure, indicate that the fluorescence intensity of the lower layer filtrate was less than 5% of the fluorescence intensity of the original oligomeric small molecule nanoparticle aqueous solution, indicating good sealing efficiency.
[0132] This invention characterizes fluorescence intensity at the maximum absorption wavelength. 1 mL of the corresponding fluorescent oligomeric nanoparticle probe aqueous solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 1000 g for 3 minutes. The lower filtrate was collected and measured on a Shimadzu-RF-5301PC fluorescence spectrometer. The fluorescence intensity results for the fluorescent oligomeric nanoparticle probes of Examples 5, 8-11, and Comparative Examples 1-3 are as follows: Figure 8As shown, with the fluorescence intensity of Example 5 as 1, the fluorescence intensities of other examples and comparative examples are measured relative to the fluorescence intensity of Example 5. In the preparation of fluorescent oligomeric nanoparticle probes, the present invention first prepares compound W-2 by reacting 2-bromofluorene and fluorene-2,7-diboron pinacol ester. Then, it reacts with different compounds to prepare oligomeric molecules of different colors. Compound W-2 reacts with 2,7-dibromofluorene to prepare a blue oligomeric molecule, compound W-2 reacts with 4,7-dibromo-2,1,3-benzothiadiazole to prepare a yellow oligomeric molecule, and compound W-2 reacts with 4... A red oligomer was prepared by reacting 7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole. Then, different colored oligomers were mixed with a silicon-oxygen precursor and a functional polymer to prepare nanoparticles via nanoprecipitation. The nanoparticles were then encapsulated with a silica shell and functionalized to prepare fluorescent oligomer nanoparticle probes. The silicon-oxygen precursor was TMOS, the functional polymer was PSMA, the silica shell was prepared from activated silica, and the functionalizing reagent included sodium carboxyethylsilanetriol. Blue fluorescent oligomers could also be prepared using the above methods. Photopolymer nanoparticle probes, yellow fluorescent oligoparticle probes, and red fluorescent oligoparticle probes were used. The blue fluorescent oligoparticle probe from Example 5 was used as a representative, with its fluorescence intensity in aqueous solution set as 1. The relative fluorescence intensity of the blue fluorescent oligoparticle probes in Examples 8-11 and Comparative Examples 1-3 in aqueous solution relative to Example 5 was used to characterize the sealing performance. The lower the relative fluorescence intensity, the less oligoparticle leakage into the solution, and therefore the better the sealing performance. When using sodium carboxyethylsilane triol, 3-aminopropyltrihydroxysilane can also be added. A fluorescent oligomeric nanoparticle probe prepared from sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane exhibits good sealing properties. Both sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane show good sealing effects within suitable usage ranges. However, when the amount of either sodium carboxyethylsilanetriol or 3-aminopropyltrihydroxysilane used is low, the other, even within a reasonable usage range, fails to effectively improve the sealing effect on the oligomeric small molecules in the fluorescent oligomeric nanoparticle probe. Furthermore, when the amounts of both sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane are low, the effect remains unsatisfactory. This invention also reveals that adding 1,1,3,3-tetramethyl-1,3-disiloxanediol as a functional agent can further improve the sealing effect on the oligomeric small molecules in the fluorescent oligomeric nanoparticle probe.
[0133] This invention explores the application of the fluorescent oligomeric nanoparticle probes prepared in Examples 5-7 in confocal imaging. Cellular microtubules were labeled using fluorescent oligomeric nanoparticle probes of three colors, and imaging was performed using confocal imaging. The steps are as follows:
[0134] (1) African green monkey kidney cells BS-C-1 were selected and seeded in confocal microplates at a density of 3 × 10⁶ cells per well. 4 One cell was cultured overnight at 37°C and 5% CO2 to allow the cells to adhere to the culture dish; the culture medium for BS-C-1 cells was aspirated from the culture dish and the cells were washed three times with PBS buffer solution.
[0135] (2) Add a small amount of extract to the culture dish, just enough to cover the cell surface; after 3 minutes, aspirate the extract and rinse 3 times with PBS buffer solution; the extract contains 1 mM EGTA, 1 mM MgCl2, 0.2 wt% Triton X-100 and 0.1 MPIPES.
[0136] (3) Add 200 μL of fixative to each well and let stand for 15 min. Then remove the fixative and rinse three times with PBS buffer solution. The fixative contains 4% PFA and 0.1 wt% GA.
[0137] (4) Add 200 μL of drilling solution to each well and let stand for 5 min. Then, remove the drilling solution and rinse 3 times with PBS buffer solution. The drilling solution contains 0.5 wt% Triton X-100.
[0138] (5) Add 1 mL of blocking solution, let stand for 30 min, then aspirate and wash with PBS buffer solution; the blocking solution contains 5 wt% BSA and 0.5 wt% Triton X-100 PBS solution.
[0139] (6) Dilute the primary antibody Anti-beta Tubulin (Abcam, cat. no. Ab179513) solution at a ratio of 1:200 in the blocking solution, add 100 μL of the primary antibody dilution solution to the culture dish and gently shake on a shaker for 60 min, and wash 3 times with PBS buffer solution.
[0140] (7) Dilute the secondary antibody Goat Polyclonal Antibody to Rabbit IgG (H&L) - Biotin (Yuantai Bio, cat. no. P50075) solution at a ratio of 1:200 in the blocking solution, add 100 μL of primary antibody dilution solution to the culture dish and gently shake on a shaker for 60 min, and wash 3 times with PBS buffer solution.
[0141] (8) The fluorescent oligomeric nanoparticle probe was coupled with streptavidin (SA) for 4 h under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and the free streptavidin was removed by ultrafiltration. The fluorescent oligomeric nanoparticle probes were selected from those prepared in Examples 5-7.
[0142] (9) After step (7), add streptavidin-modified fluorescent oligomer nanoparticle probes, gently shake for 60 min; rinse thoroughly with PBS buffer solution, and store at 4°C.
[0143] (10) Confocal imaging: Using CSU-W1-SoRa, excitation light at 405nm, 488nm and 561nm was used to perform confocal imaging of cells.
[0144] (11) Super-resolution imaging: Super-resolution imaging was performed using STED.
[0145] The coupling steps for streptavidin-modified fluorescent oligomer nanoparticle probes are as follows:
[0146] (1) Buffer and stock solution: Fluorescent oligomeric nanoparticle probe aqueous solution (approximately 22 nM), PES buffer (1M, pH 7.3), polyethylene glycol (PEG, MW 3350) in MilliQ water (5 wt%), bovine serum albumin (BSA, 0 wt%) in 20 mM HEPES, and coupling wash (50 mg PEG + 49 mL MilliQ water + 1 mL HEPES).
[0147] (2) Add 4 mL of fluorescent oligomeric nanoparticle probe aqueous solution to a centrifuge tube, and then add the following solutions in sequence: 80 μL of PEG (5 wt%), vortex mix; 80 μL of concentrated HEPES buffer (1 M), vortex mix; 240 μL of streptavidin (1 mg / mL), vortex mix to obtain fluorescent oligomeric nanoparticle probe mixture.
[0148] (3) Prepare a fresh EDC solution (5 mg / mL) in MilliQ water, and then add 80 μL of the EDC solution to the fluorescent oligomeric nanoparticle probe mixture. Vortex mix. Then stir at room temperature for 4 hours.
[0149] (4) Four hours after the bio-coupling reaction, 80 μL of BSA (10 wt%) was added to the solution and allowed to continue for 20 minutes to obtain a mixture of nanoparticles and streptavidin.
[0150] (5) Transfer the nanoparticle-streptavidin mixture to a centrifugal ultrafiltration tube (Amicon® Ultra-4, MWCO: 100kDa) and concentrate it to 0.5 mL using a centrifuge. Then add the final volume to mL with coupling wash buffer and repeat the ultrafiltration 6 times to obtain purified streptavidin-modified fluorescent oligomer nanoparticle probes.
[0151] The results are as follows Figure 9 As shown, a) is a confocal imaging of cell microtubules by streptavidin-oligomeric nanoparticle conjugate prepared with blue fluorescent oligomeric nanoparticle probes; b) is a confocal imaging of cell microtubules by streptavidin-oligomeric nanoparticle conjugate prepared with yellow fluorescent oligomeric nanoparticle probes; and c) is a confocal imaging of cell microtubules by streptavidin-oligomeric nanoparticle conjugate prepared with red fluorescent oligomeric nanoparticle probes. All three colors of fluorescent oligomeric nanoparticle probes show relatively continuous imaging effects on cell microtubules.
[0152] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0153] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method for preparing a fluorescent oligomeric nanoparticle probe, comprising: Compound W-2 was prepared by reacting 2-bromofluorene with pinacol fluorene-2,7-diborate; the molar amount of pinacol fluorene-2,7-diborate used was 50-200% of the molar amount of 2-bromofluorene. Oligomeric small molecules were prepared by reacting compound W-2 with a dibromine reagent; Oligomeric small molecules were added to tetrahydrofuran and mixed to obtain an oligomeric small molecule solution of 0.5-2 mg / mL; then functional polymers were added to tetrahydrofuran and mixed to obtain a PSMA solution of 0.5-2 mg / mL; silicon oxide precursor TMOS was added to tetrahydrofuran and mixed to obtain a TMOS solution of 0.5-2 mg / mL; TEOS was added to tetrahydrofuran and mixed to obtain a TEOS solution of 0.5-2 mg / mL; the oligomeric small molecule solution, PSMA solution, TMOS solution and TEOS solution were added to tetrahydrofuran and mixed to obtain a mixed solution; ammonia solution was added to an ultrasonic water bath and ultrasonicated for 1-5 min; then the solution was concentrated by nitrogen blowing at 80-100℃; the solution was filtered through a 220 nm filter while hot; then activated silica was added and stirred for 6-24 h; then functionalizing reagents were added and stirred for 2-8 h for functionalization treatment to prepare fluorescent oligomeric nanoparticle probes; The dibromo reagent is at least one of 2,7-dibromofluorene, 4,7-dibromo-2,1,3-benzothiadiazole, and 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole; The functional polymer is PSMA; The silicon-oxygen precursors are TMOS and TEOS; The functionalizing reagents are sodium carboxyethylsilanetriol and 3-aminopropyltrihydroxysilane; the amount of sodium carboxyethylsilanetriol used is 0.2-1 wt% of the mixed solution; the amount of 3-aminopropyltrihydroxysilane used is 0.1-0.6 wt% of the mixed solution. The volume of the oligomer small molecule solution used in the mixed solution is 10-20% of the volume of tetrahydrofuran used. The volume of PSMA solution used in the mixed solution is 50-200% of the volume of oligomeric small molecule solution used; the volume of TMOS solution used in the mixed solution is 50-200% of the volume of oligomeric small molecule solution used; and the volume of TEOS solution used in the mixed solution is 50-200% of the volume of oligomeric small molecule solution used.
2. The preparation method according to claim 1, characterized in that, The activated silica is prepared from sodium silicate solution through a hydrogen-form cation exchange resin.
3. The preparation method according to claim 1, characterized in that, When the dibromo reagent is 2,7-dibromofluorene, the small oligomer molecule is a blue small oligomer molecule B-1.
4. The preparation method according to claim 1, characterized in that, When the dibromo reagent is 4,7-dibromo-2,1,3-benzothiadiazole, the small oligomer molecule is a yellow small oligomer molecule Y-1.
5. The preparation method according to claim 1, characterized in that, When the dibromo reagent is 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole, the small oligomer molecule is a yellow small oligomer molecule R-1.
6. The preparation method according to claim 1, characterized in that, The functionalizing agent further includes 1,1,3,3-tetramethyl-1,3-disiloxanediol, wherein the amount of 1,1,3,3-tetramethyl-1,3-disiloxanediol used is 0.05-0.3 wt% of the mixed solution.
7. Fluorescent oligomeric nanoparticle probes prepared by the method according to any one of claims 1-6.