Ultra-small-size high-brightness nano probe and single protein tracking application thereof

Abstract

The invention provides an ultra-small-size high-brightness nanoprobe and a single protein tracking application thereof, the nanodot is of a core-shell structure, and the core-shell structure comprises: a fluorescent core, which is formed by a single-chain semiconductor polymer; the shell layer is formed by an amphiphilic compound, a hydrophobic part of the amphiphilic compound is combined with the semiconductor polymer, a hydrophilic group is located on the surface of the shell layer to serve as an active group, and the average particle size of the nanodot is smaller than 5 nm. The single-chain ultra-small polymer point provided by the invention can realize high temporal-spatial resolution tracking of a single motor protein in a living cell, and can accurately analyze the motion step length of about 16 nm.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to an ultra-small, high-brightness nanoprobe and its single-protein tracking application. Background Technology

[0002] Single-particle tracking imaging in live cells enables the tracking of the dynamic changes of individual fluorescent molecules over time and space at high spatiotemporal resolution. This allows for real-time in-situ observation and statistical analysis of biomolecules in different states, making it a powerful tool for studying the distribution, structure, dynamics, and precise movement of biomolecules in living cells and providing an effective pathway for elucidating life processes (Nat. Chem. Biol. 2014, 10, 524-532). Fluorescent probes, as "signal sources," are indispensable in fluorescence imaging. However, while traditional fluorescent proteins and dyes have the advantages of small size and ease of labeling within living cells, their brightness and photostability are poor, often making it difficult to resolve the motion behavior and functional mechanisms of individual protein molecules at high spatiotemporal resolution (Chem. Soc. Rev. 2009, 38, 2887-2921; Chem. Soc. Rev. 2023, 52, 5607-5651). In recent years, fluorescent nanoparticles have become star probes in fluorescence imaging due to their bright fluorescence and excellent photostability, allowing for long-term single-particle tracking imaging. Examples include metal quantum dots, upconversion nanoparticles, and AIE nanoparticles (Nat. Methods. 2018, 15, 415-423). However, the generally large particle size (>10 nm) of metal nanoparticles and the complexity of the interface caused by the core-shell coating required for stability in aqueous solutions limit their ability to specifically label single proteins in living cells without interfering with their physiological behavior for tracking imaging (Chem. Soc. Rev. 2015, 44, 4792-4834). Therefore, the development of nanoprobes with ultra-small size, clear interfaces, and ultra-high brightness holds promise for solving the problem of undisturbed, high spatiotemporal resolution dynamic tracking imaging of specific target proteins in living cells, thereby elucidating the molecular mechanisms in complex biological systems. Summary of the Invention

[0003] This invention addresses the key problem of insufficient spatiotemporal resolution or interference with protein physiological behavior encountered by traditional fluorescent probes when labeling single proteins in living cells for high spatiotemporal resolution tracking imaging. This invention prepares ultrasmall nanodots (<5 nm) composed of single polymer chains, exhibiting clear composition and ultra-small size, excellent brightness, and ease of biofunctionalization. By modifying with functional small molecules such as HALO / SNAP-Tag, it can specifically target functional protein molecules expressing corresponding tagged proteins within living cells.

[0004] In one aspect, the present invention provides a nanodot, wherein the nanodot has a core-shell structure, and the core-shell structure comprises:

[0005] Fluorescent core: composed of single-chain semiconductor polymer;

[0006] Shell: Composed of an amphiphilic polymer, wherein the hydrophobic groups of the amphiphilic polymer are combined with the semiconductor polymer, and the hydrophilic groups are located on the surface of the shell as active groups, and the average particle size of the nanodots is <5nm.

[0007] In one embodiment of the present invention, the semiconductor polymer is selected from conjugated polymers, polythiophene, polyfluorene, polystyrene, polyphenylacetylene, and their derivatives. In another embodiment of the present invention, the semiconductor polymer is selected from CNPPV, PDFDP, or a combination thereof.

[0008] In one embodiment of the present invention, the amphiphilic polymer includes a hydrophobic group and a hydrophilic group, wherein the hydrophilic group is selected from carboxyl, amino, or hydroxyl groups. In another embodiment of the present invention, the amphiphilic compound is selected from polystyrene maleic anhydride.

[0009] In one embodiment of the present invention, the nanodots have a glassy structure.

[0010] In one embodiment of the present invention, the nanodots are prepared by a glass freezing method. In another embodiment, the semiconductor polymer and the amphiphilic polymer are mixed and dissolved, then rapidly cooled in a low-temperature environment to transform them into a glassy state, thereby obtaining the nanodots.

[0011] In one embodiment of the present invention, the rapid cooling rate is greater than 1000°C / min.

[0012] In one embodiment of the present invention, the nanodots further include modification. In one embodiment of the present invention, the modifying group is covalently coupled to the nanodots via an active group. In one embodiment of the present invention, the modifying group includes antibodies, antibody fragments, ligands, receptors, peptides, proteins, nucleic acids, aptamers, carbohydrates, drug molecules, small molecule compounds, fluorescent probes, radiolabels, enzymes, signaling molecules, polymeric modifying groups, or magnetic / functional nanoparticles. In one embodiment of the present invention, the modifying group is a ligand. Preferably, the ligand is a HaloTag ligand.

[0013] In one embodiment of the present invention, the nanodots have improved photostability and resistance to photobleaching.

[0014] In one embodiment of the present invention, the nanodots can be used for bioimaging or drug delivery.

[0015] In a second aspect, the present invention provides a method for preparing nanodots by means of a glass freezing method.

[0016] In one embodiment of the present invention, the method includes the following steps:

[0017] (a) Dissolve the semiconductor polymer and the amphiphilic polymer in a solvent to form a mixed solution;

[0018] (b) The mixed solution is placed in a low-temperature environment for rapid cooling to transform it into a glassy state;

[0019] (c) Remove the solvent to obtain the vitrified nanodot product.

[0020] In one embodiment of the present invention, the solvent is tetrahydrofuran, dioxane, dichloromethane, or a combination thereof. Exemplarily, in one embodiment of the present invention, the solvent is a mixed solvent of tetrahydrofuran and dioxane, with a volume ratio of tetrahydrofuran to dioxane of 2:8.

[0021] In one embodiment of the present invention, the concentration of the semiconductor polymer is 0.1-2 mg / mL, such as 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, or 2 mg / mL; the concentration of the amphiphilic compound is 10-100 mg / mL, such as 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL. The volume ratio of the semiconductor polymer to the amphiphilic compound is 1:1-1:10, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0022] In one embodiment of the present invention, the rapid cooling rate is greater than 1000°C / min.

[0023] In one embodiment of the present invention, the low temperature is the liquid nitrogen temperature.

[0024] In one embodiment of the present invention, the mixed solution is slowly dripped from a certain height onto a freezing stage at liquid nitrogen temperature, causing it to rapidly solidify at low temperature and form a glassy structure. In another embodiment of the present invention, the mixed solution is slowly dripped from a height of 0.5m onto a freezing stage at liquid nitrogen temperature.

[0025] In one embodiment of the present invention, the freezing stage is a glass freezing stage.

[0026] In one embodiment of the present invention, the solvent is removed by freeze drying.

[0027] In one embodiment of the present invention, the obtained dried powder is hydrolyzed and / or dispersed in an aqueous solution (such as an alkaline solution) to obtain single-chain ultrasmall polymer nanodots stably dispersed in the aqueous solution. In the aqueous solution, the single-chain polymer forms stably dispersed single-chain ultrasmall polymer nanodots through intramolecular entanglement.

[0028] In one embodiment of the present invention, the method further includes the steps of centrifugation, filtration, and ultrafiltration purification.

[0029] In one embodiment of the present invention, the method further includes modifying the nanodots.

[0030] In one embodiment of the present invention, the modifying group is covalently coupled to the nanodots via an active group. In another embodiment of the present invention, the modifying group is covalently coupled to the carboxyl group of the nanodots under the action of an activator. In one embodiment of the present invention, the activator is 5 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 5 mg / mL).

[0031] In one embodiment of the present invention, the modifying group includes antibodies, antibody fragments, ligands, receptors, peptides, proteins, nucleic acids, aptamers, carbohydrates, drug molecules, small molecule compounds, fluorescent probes, radiolabels, enzymes, signaling molecules, polymer modifying groups, or magnetic / functional nanoparticles. In one embodiment of the present invention, the modifying group is a ligand. Preferably, the ligand is a HaloTag ligand.

[0032] In a third aspect, the present invention provides a nanoprobe, kit, or composition comprising the aforementioned nanodots.

[0033] In a fourth aspect, the present invention provides the application of the above-mentioned nanodots, nanoprobes, kits or compositions in bioimaging and targeted drug delivery.

[0034] In one embodiment of the present invention, the ultra-small nanodots decay slowly and have stronger photostability during long-term imaging.

[0035] In one embodiment of the present invention, the ultrasmall polymer nanodots possess single-molecule resolution. In another embodiment, the ultrasmall polymer nanodots can achieve signal detection at the single-molecule level, enabling high-sensitivity analysis or characterization of the chemical or biological function of a single molecule. In yet another embodiment, the nanodots can be used for high spatiotemporal resolution tracking of single motor proteins within living cells; furthermore, the nanodots can be resolved to have a motion step size of approximately 16 nm.

[0036] In a fifth aspect, the present invention provides a detection method comprising using the above-described nanodots, nanoprobes, kits, or compositions for detection.

[0037] In one embodiment of the present invention, the detection method includes using the aforementioned nanodots to track the motion characteristics of individual proteins. In another embodiment of the present invention, the detection method includes detecting the step length of a single motor protein within a living cell.

[0038] In one detection method of the present invention, the method includes introducing ultrasmall polymer nanodots with functional ligand small molecules modified on their surface into living cells expressing target protein particles for labeling, collecting the fluorescence signal of the labeled protein using confocal microscopy, and detecting and analyzing the protein's motion trajectory using a localization algorithm and trajectory analysis software. In one embodiment of the present invention, the ligand small molecule is a HaloTag ligand, and the target protein is a HaloTag-labeled kinesin protein.

[0039] Compared to polymer nanoparticles prepared by traditional nanoprecipitation methods, the single-chain ultrasmall polymer dots of this invention exhibit clear composition and ultra-small size, while also possessing excellent brightness. Leveraging this high brightness, high spatiotemporal resolution tracking of individual motor proteins within living cells can be achieved using only a standard rotating confocal fluorescence microscope, precisely resolving their motion step length of approximately 16 nm. This marks the first time that the step length of a single motor protein within living cells has been resolved through an innovative fluorescent probe without relying on complex and expensive equipment (such as MINFLUX). Attached Figure Description

[0040] Figure 1 A schematic diagram illustrating the principle of preparing ultra-small, ultra-high brightness nanodots using vitrification freezing technology.

[0041] Figure 2 Size characterization of ultrasmall polymer nanodots

[0042] Figure 3 Brightness characterization of ultrasmall polymer nanodots

[0043] Figure 4 A schematic diagram and imaging results of labeling individual motor proteins with ultra-small nanodots.

[0044] Figure 5 The effects of ultrasmall nanodots and commercial dyes on the motor protein motility and the photostability of ultrasmall nanodots and commercial dyes were investigated.

[0045] Figure 6 (a) High spatiotemporal resolution (50Hz) display of ultra-small nanodots to analyze the stepping motion of individual motor proteins. (b) Local magnification of the stepping trajectory of a single motor protein; actual data (black line), fitted data (red line), red arrows indicate the individual step size of the motor protein. (c) Gaussian fit to a normal distribution for all step sizes. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0047] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-pheny-lene)], ADS 110 RE), PDFDP(poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}], ADS 111 RE)

[0048] The single-chain ultrasmall polymer nanodots of the present invention refer to ultrasmall nanoparticles formed by the coiling of a single semiconductor fluorescent polymer chain (or single-chain semiconductor fluorescent polymer). The polymer chain exists in the form of a single molecule, rather than a composite structure formed by the aggregation or multi-chain entanglement of multiple polymer chains. It has a clearer internal composition and higher operability of nanoparticle surface chemistry.

[0049] Example 1: Preparation of single-chain ultrasmall polymer nanodots

[0050] The vitrification freezing technique was used to prepare nanodots with ultra-small size and ultra-high brightness. The specific steps are as follows:

[0051] 200 μL of a semiconductor polymer chain solution (1 mg / mL) and 200 μL (50 mg / mL) of styrene-maleic anhydride were injected into a mixed solvent (tetrahydrofuran / dioxane = 2:8). The mixture was thoroughly mixed in an ultrasonic apparatus. The mixture was then slowly dripped from a height of 0.5 m into a clean glass dish pre-cooled with liquid nitrogen, allowing it to solidify rapidly at low temperature. The solvent was further removed by freeze-drying. The resulting dried powder was hydrolyzed and dispersed in an alkaline solution. Finally, the powder was purified by centrifugation, filtration, and ultrafiltration to obtain stable single-chain ultrasmall polymer nanodots CNPPVPdots and PDFDPdots dispersed in an aqueous solution. A schematic diagram of the principle of vitrification freezing technology for preparing ultrasmall, ultra-high brightness nanodots is shown below. Figure 1As shown. The size of the Pdot was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results are as follows. Figure 2 As shown in the figure, the average diameter of both CNPPV Pdot and PDFDP Pdot is around 5 nm.

[0052] Using EGFP, Alexa 488, and QDs 605 as controls, photon counting was performed by detecting fluorescence signals under a single-molecule fluorescence microscope. The results are as follows: Figure 3 As shown, CNPPV Pdot and PDFDP Pdot have the highest photon emission, with photon counts reaching 60,000-80,000, far exceeding other groups, indicating that these two types of materials have stronger luminescence capabilities.

[0053] Example 2: Small molecule modification of ultra-small polymer dot functionalized tags

[0054] 150 μL of the ultra-small polymer nanodot solution was injected into a 10 mL glass bottle containing 800 μL of ultrapure water. Then, 20 μL of (N-hydroxyethylpiperazine-2-ethanesulfonic acid (1 M), HaloTag ligand molecule (5 μL, 5 mg / mL) and (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5 μL, 5 mg / mL) were added and mixed thoroughly. The mixture was then stirred at room temperature for 2 hours. Subsequently, it was purified using a 30 kDa ultrafiltration centrifuge tube to remove excess solute and stored at 4 °C in the dark for further use.

[0055] Example 3: Labeling of single kinesin with ultrasmall polymer dots-functional tags

[0056] To specifically label a single intracellular protein (using kinesin as an example), a HaloTag-Kinesin plasmid (2.2 μg / mL, 0.2 μL) was constructed. Ultra-small polymer dots, modified with HaloTag ligands (0.2 μL), were introduced into live cells expressing the HaloTag-Kinesin plasmid using electroporation to specifically label a single Kinesin protein tagged with HaloTag. The cells were then incubated overnight to allow for full recovery and preparation for subsequent imaging.

[0057] Example 4: High spatiotemporal resolution tracking imaging of a single Kinesin protein

[0058] Fluorescence signals of the ultrasmall nanoprobes were collected using a standard rotating confocal microscope (CSU-W1 scanning unit, excitation wavelength 488nm, 100x oil immersion objective, bandpass filter 600 / 52, exposure time 20ms). Subpixel-level localization was achieved using the open-source software TrackMate 7 combined with the LoG (Laplacian Gaussian fitting) algorithm. To analyze the motion characteristics and trajectory behavior of individual Kinesin proteins, the trajectory displacement of individual Kinesin proteins was obtained using TrackMate software, and the displacement trajectory of the motor protein was fitted using an open-source step detection program to obtain its single-step length (…). Figure 6 ).

[0059] A schematic diagram of the principle and imaging results of ultra-small nanodot labeling of single motor proteins are shown below. Figure 4 As shown.

[0060] The effects of ultrasmall nanodots and commercial dyes on the motor protein motility and the photostability of ultrasmall nanodots and commercial dyes, such as Figure 5 As shown in the results, both ultrasmall nanodots and commercial dyes labeled the motor proteins, allowing the motor proteins to maintain strong random Brownian motion. This indicates that the labeling has minimal interference with the motor proteins' directional stepping motion (such as advancement along microtubules), and their motion behavior is close to their natural state. Compared to commercial dyes, ultrasmall nanodots decay more slowly (>10 seconds) and exhibit stronger photostability during long-term imaging.

[0061] from Figure 6 It can be seen that, Figure 6 The image, with high spatiotemporal resolution (50 Hz acquisition frequency), displays the position change curve of a single motor protein over time, exhibiting a typical step-like ascending pattern, indicating that the motor protein undergoes process-oriented transport along microtubules. The high resolution allows for the capture of subtle fluctuations (such as noise or brief pauses), demonstrating the effectiveness of ultrasmall polymer nanodots at the single-molecule level without significantly interfering with natural motion dynamics. Figure 6In Figure b, the single step size marked by the red arrow is approximately 16 nm, which is consistent with the standard step size of kinesin. The timescales are labeled 500 ms (overall) and 100 ms (details), indicating that rapid stepping (~100 ms / step) can be clearly distinguished at high temporal resolution, verifying the dye's high photostability and low photobleaching properties, supporting long-term single-molecule tracking without signal loss. Figure c shows the normal distribution of all step sizes through histograms and Gaussian fitting, with an average step size of approximately 15.7 nm, mainly concentrated in the 10-20 nm range. The good Gaussian fitting (narrow peaks) indicates high data consistency and small step size variation, further confirming the reliability of the method. Using ultrasmall nanodots, the stepping motion of a single motor protein was successfully captured at high spatiotemporal resolution, with a step size of ~16 nm, consistent with the reported kinesin step size in the literature. The movement speed can be estimated as ~160 nm / s (based on step size and timescale).

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although modifications or equivalent substitutions are made with reference to preferred embodiments, they do not depart from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nanodot, characterized in that, The nanodot is a core-shell structure, the core-shell structure comprising: a fluorescent core consisting of a single-stranded semiconductor polymer; a shell layer consisting of an amphiphilic polymer, the hydrophobic group of the amphiphilic polymer binding with the semiconductor polymer, and the hydrophilic group on the surface of the shell layer as an active group, the average particle size of the nanodot being < 5 nm.

2. A nanodot according to claim 1, wherein, The semiconductor polymer is selected from conjugated polymers, polythiophene, polyfluorene, polystyrene, polyphenylacetylene and derivatives thereof; alternatively, the semiconductor polymer is selected from CNPPV, PDFDP or a combination thereof; and / or the amphiphilic polymer comprises a hydrophobic group and a hydrophilic group, the hydrophilic group being selected from carboxyl, amino or hydroxyl; alternatively, the amphiphilic compound is selected from polystyrene maleic anhydride.

3. The nanodot of claim 1, wherein the nanodot is a semiconductor nanodot. The nanodot is a glassy structure.

4. The nanodot of claim 1, wherein the nanodot is a semiconductor nanodot. The nanodot is prepared by a glass freezing method; Alternatively, the semiconductor polymer and the amphiphilic polymer are mixed and dissolved, and are placed in a low temperature environment for rapid cooling to convert them into a glassy state to obtain the nanodot; alternatively, the cooling rate of the rapid cooling is greater than 1000℃ / min.

5. The nanodot of claim 1, wherein the nanodot is a semiconductor nanodot. The nanodot further comprises a modification; Alternatively, the modification group is covalently coupled to the nanodot through an active group; alternatively, the modification group comprises an antibody, an antibody fragment, a ligand, a receptor, a peptide, a protein, a nucleic acid, an aptamer, a saccharide, a drug molecule, a small molecule compound, a fluorescent probe, a radioactive label, an enzyme, a signal molecule, a polymer modification group or a magnetic / functional nanoparticle; alternatively, the modification group is a ligand; preferably, the ligand is a HaloTag ligand.

6. A method of preparing nanodots, comprising: The nanodot is prepared by a glass freezing method; Alternatively, the method comprises the following steps: (a) dissolving the semiconductor polymer and the amphiphilic polymer in a solvent to form a mixed solution; (b) placing the mixed solution in a low temperature environment for rapid cooling to convert them into a glassy state; (c) removing the solvent to obtain a glassy nanodot product.

7. The method for preparing nanodots as described in claim 6, characterized in that, The solvent is tetrahydrofuran, dioxane, dichloromethane or a combination thereof; and / or the concentration of the semiconductor polymer is 0.1-2 mg / mL; and / or the concentration of the amphiphilic compound is 10-100 mg / mL; and / or the volume ratio of the semiconductor polymer to the amphiphilic compound is 1:1-1:10; and / or the cooling rate of the rapid cooling is greater than 1000℃ / min; and / or the low temperature is liquid nitrogen temperature; and / or the mixed solution is slowly dropped from a certain height onto a freezing platform at liquid nitrogen temperature to rapidly solidify at low temperature to form a glassy structure; and / or the solvent is removed by freeze-drying; and / or the obtained dry powder is hydrolyzed and / or dispersed in an aqueous solution to obtain a single-stranded ultrasmall polymer nanodot; wherein, in the aqueous solution, the single-stranded polymer is wound intramolecularly to form a single-stranded ultrasmall polymer nanodot; and / or the method further comprises the steps of centrifugation, filtration and ultrafiltration purification; and / or the method further comprises modifying the nanodot.

8. A nanoprobe, kit or composition comprising the nanodot of any one of claims 1-5.

9. Use of the nanodots of any one of claims 1-5, the nanoprobe, the kit or the composition of claim 8 in bioimaging, targeted drug delivery. Optionally, the ultrasmall polymer nanodots can achieve signal detection at the single molecule level, for high sensitivity analysis or characterization of single molecule chemical or biological functions. Optionally, the ultrasmall polymer nanodots have single molecule resolution capability; preferably the nanodots can be used for resolution tracking of single motor proteins in living cells.

10. A detection method comprising using the nanodots of any one of claims 1-5, the nanoprobe, the kit or the composition of claim 8 for detection.

Images