Gold homodimer AuHDs probe and its synthesis method and its application in biological dynamic analysis

Abstract

The present invention discloses a gold homodimer (AuHDs) probe based on anisotropic scattering properties and uniform surface chemical properties, a preparation method and an application thereof. The present invention also discloses a preparation method of the AuHDs probe obtained by a stepwise growth method and DNA modification and purification, wherein the synthesized probe has uniform physical and chemical properties and dumbbell-shaped anisotropic scattering properties. The present invention also discloses a single particle tracking (SPT) method assisted by a colorimetric algorithm (CBA), which can be used to accurately track the rotational motion of the AuHDs probe under a dark field microscope (DFM). The present invention brings new inspiration to the development of anisotropic imaging probes in various cellular processes and the dynamics study of vesicle movement during endocytosis, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials and biodynamic analysis, and specifically relates to a novel anisotropic nanoprobe gold homodimer AuHDs and a synthesis method thereof, as well as an application thereof in biodynamic analysis. Background Art

[0002] Vesicle motility plays a key role in many important cellular processes, such as phagocytosis, cell communication, cell signaling, cell migration and division, and receptor-mediated endocytosis. However, the link between the interaction rotation driven by the nanomechanical scaffold and the detachment of the nascent vesicle during vesicle motility is still unclear.

[0003] To address these questions about dynamic rotation and clarify the mechanism of nanoscaffold-catalyzed motion, it is crucial to directly visualize the dynamic rotation of nascent vesicles during endocytosis at the single-particle level. Dark-field imaging (DFM) based on gold nanorods (AuNRs) is particularly important for observing the dynamic and complex behavior of nascent vesicles in vivo. However, surfactant molecules preferentially accumulate on the sides of AuNRs during growth, resulting in different chemical and crystal properties between the tip and the side, as well as strong nonspecific rotation due to the opposite charges of the side and tip. Therefore, using AuNRs as directional probes carries the risk of unexpected motion caused by surface heterogeneity, which may reduce the accuracy and fidelity of the final quantification of rotational dynamics. Summary of the invention

[0004] In order to overcome the defects of the prior art, the present invention provides a gold homodimer AuHDs probe, which is a standard dumbbell-shaped nanostructure (composed of two identical nanoparticles); and the probe has a local surface plasmon resonance (LSPR) effect and uniform and stable surface chemical properties.

[0005] Specifically, the probe is composed of two nanoparticles (NPs) of the same size, which is a dumbbell-shaped gold nanostructure in which the distance between the particles is smaller than the size of each particle; and the probe has a local surface plasmon resonance (LSPR) effect, which can present anisotropic scattering characteristics of two plasmon resonance modes; the probe also maintains the same composition, crystal structure and interface properties on the original spherical nanoparticles, so that the surface chemical properties of the probe have clear uniformity. This characteristic also leads to an unformed affinity between the probe and the membrane proteins around it, thereby reducing the unexpected movement of the probe in the endocytic vesicle.

[0006] The invention also provides a method for preparing the gold homodimer AuHDs probe.

[0007] The method is to select 32.1±0.2nm gold nanoparticles (AuNPs) as gold seeds, add a mixture of Na3Ci and HAuCl4, and grow on the above gold seeds through the "intra-particle maturation" process, thereby forming a dumbbell-shaped dimer between two attached nanoparticles.

[0008] Specifically, the AuHDs probe is synthesized by a stepwise growth method, the steps comprising:

[0009] (1) synthesizing gold nanoparticles (AuNPs) with a size of 30-32 nm and using them as the seed solution for the initial reaction;

[0010] (2) on the basis of step (1), sodium hydroxide (NaOH), sodium citrate (Na3Ci), and tetrachloroauric acid trihydrate (HAuCl43H2O) are added in sequence to react and obtain the prepared gold nano dimer;

[0011] (3) On the basis of step (2), utilizing the strong interaction of Au-S bonds, adding SH-DNA (DNA (SH-Probe2)) to prevent particle aggregation, and reacting to obtain the gold homodimer AuHDs probe;

[0012] (4) Based on the step (3), the desired dimer bands are separated by agarose gel electrophoresis reaction experiment, and the desired AuHDs probes are recovered by gel cutting.

[0013] In the step (1), HAuCl4 and Na3Ci are added in the initial stage to synthesize the precursor solution required for the reaction; the reaction temperature is 100-140°C, preferably 100°C; the reaction time is 40-60min, preferably 40min. Subsequently, HAuCl4 is continuously added to synthesize AuNPs with a size of 30-32nm, the reaction temperature is 90-100°C, preferably 90°C; the reaction time is 1-2h, preferably 2h.

[0014] In the step (2), NaOH and NaCi3 are added to the 30 nm AuNPs solution in sequence; the reaction temperature is 15-25°C, preferably 25°C; the reaction time is 10-20 min, preferably 10 min. HAuCl4 is then added again; the reaction temperature is 30-37°C, preferably 37°C; the reaction time is 30-45 min, preferably 30 min.

[0015] In the step (2), the concentrations of NaOH, Na3Ci, HAuCl43H2O and gold nanoparticles are all 25 mM;

[0016] In the step (2), the volume ratio of NaOH, Na3Ci, HAuCl43H2O and gold nanoparticles is 20-30 μL: 30-50 μL: 4-8 μL: 4-8 μL; preferably, the volume ratio is 30 μL: 40 μL: 8 μL: 8 μL.

[0017] In the step (3), DNA (SH-Probe2) is added; the ratio of DNA to gold nanodimer is 1:4000; the reaction temperature is -60 to -80°C, preferably -80°C; and the reaction time is 5 to 10 minutes, preferably 5 minutes.

[0018] In the step (4), agarose gel electrophoresis technology is used to cut the gel to recover the desired product bands to improve the yield of the AuHDs sample. The reaction voltage is 100 V, the reaction time is 30 min, and the reaction buffer is 0.5×tris-borate-EDTA (TBE), that is, Tris-boric acid with a molar concentration of 0.045 M and EDTA (TBE) with a molar concentration of 0.001 M.

[0019] The present invention also provides a gold homodimer AuHDs probe obtained by the preparation method. The probe has uniform physical and chemical properties and dumbbell-shaped anisotropic scattering characteristics; moreover, the synthesis of the gold homodimer AuHDs probe effectively avoids the problems of photobleaching of traditional fluorescent probes; more importantly, compared with other commonly used nanoprobes, the gold homodimer AuHDs probe can be synthesized without using a surfactant, which effectively avoids the toxicity of the surfactant to living cells and retains the same composition, crystal structure and interface composition as the original gold species, thereby making the surface chemical properties of the gold homodimer AuHDs probe clearly unified.

[0020] In the present invention, the agarose gel electrophoresis technology in the step (4) can greatly improve the yield and stability of the gold homodimer AuHDs probe, which greatly increases the number of the gold homodimer AuHDs probe entering the cell, thereby facilitating the accurate tracking of the rotational movement of the gold homodimer AuHDs probe under a dark field microscope (DFM).

[0021] The present invention also proposes the application of the gold homodimer AuHDs probe in the imaging mode under dark field microscopy, in the spectral characteristics under dark field microscopy (DFM) and ultraviolet-visible light, in the precise tracking of the cell endocytic movement under dark field microscopy (DFM), and in the precise tracking of the rotational movement of cargo vesicles during the endocytic process under dark field microscopy (DFM).

[0022] The present invention also provides an imaging mode of the probe under a dark field microscope (DFM).

[0023] The invention also provides the spectral characteristics of the probe under dark field microscopy (DFM) and ultraviolet-visible light.

[0024] The present invention also provides the cell endocytosis movement process of the probe under a dark field microscope (DFM).

[0025] The present invention also provides the use of the probe in monitoring the rotational movement of cargo vesicles in the process of cell transmembrane under a dark field microscope (DFM).

[0026] The present invention also provides a method for applying the gold homodimer AuHDs probe to an imaging mode of a dark field microscope (DFM), the method comprising:

[0027] (1) The AuHDs solution is titrated on a silanized glass slide and optical imaging is performed using a dark field microscope;

[0028] (2) The R and G scattering intensities of AuHDs under dark field were measured by CBA algorithm, and the polar angle (φ) of AuHDs was calculated according to the dual-channel differential equation: f(RG) = (1.36sin 2 φ-0.36sin 4 φ);

[0029] The silanization treatment time used in the method is 20-30 minutes, preferably 30 minutes.

[0030] The exposure time of the imaging mode used in the method is 80-100 ms, preferably 100 ms.

[0031] In a specific embodiment, the AuHDs probe is applied to a dark field microscope (DFM) imaging mode method, comprising:

[0032] (1) Producing polarization images:

[0033] The AuHDs probe (PBS treated) was titrated on a silanized glass slide and observed under a dark field microscope after 30 minutes. The dark field microscope polarizer was adjusted every 3° to record the change of the dark field color of AuHDs with different polar angles (φ).

[0034] (2) Preparation of standard curve:

[0035] The AuHDs probe solution [PBS treatment and cMEM (complete medium) treatment] was titrated on a silanized slide and placed under a dark field microscope for observation after 30 minutes. The polarizer of the dark field microscope was adjusted every 3°, and the intensity change value of the RG of the AuHDs probe at different polar angles (φ) was recorded. Then, the change curve of f(RG) and polar angle was drawn to obtain the relationship between f(RG) and sin 2 Proportional to φ.

[0036] The present invention also provides a method for applying the AuHDs probe to spectral characteristics under dark field microscopy (DFM) and UV-visible spectroscopy, the method comprising:

[0037] (1) The AuHDs solution is titrated onto a silanized glass slide, and the scattering spectrum is photographed using a dark field microscope;

[0038] (2) The AuHDs solution is diluted to a certain concentration, and a certain volume of sample is taken for UV-visible light absorption test.

[0039] Wherein, the silanization treatment time used in the method is 20-30 minutes, preferably 30 minutes.

[0040] The exposure time of the imaging mode used in the method is 80-100 ms, preferably 100 ms.

[0041] Wherein, the sample concentration used in the method is 0.5-1.0nM, preferably 1.0nM.

[0042] The sample volume used in the method is 200-500 μL, preferably 500 μL.

[0043] In a specific embodiment, the AuHDs probe is applied to a spectral characterization method under dark field microscopy (DFM) and UV-visible spectroscopy, comprising:

[0044] (1) Create a scattering curve:

[0045] The AuHDs probe (PBS treated) was titrated on a silanized glass slide and observed under a dark field microscope after 30 minutes. The scattered light signal of the AuHDs probe was collected by a dark field microscope to draw a scattering curve image.

[0046] (2) Preparation of UV-Visible spectral curve:

[0047] The AuHDs probe is placed in a dedicated UV test dish, the UV signal of the AuHDs probe is collected by UV-visible absorption spectroscopy, and a UV curve image is drawn.

[0048] The present invention also provides a method for applying the AuHDs probe to a cell internalization process under dark field microscopy (DFM), the method comprising:

[0049] (1) co-culturing the AuHDs probe solution with cells to allow the AuHDs probe to enter the cells;

[0050] (2) The cellular interactions of the AuHDs during endocytosis were analyzed using various endocytosis inhibitors, including chlorpromazine (CPZ), filipin III, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), and it was determined that the AuHDs can be used to monitor the movement of cargo vesicles during endocytosis.

[0051] The AuHDs probe concentration used in the method is 0.05 nM, the number of cells is 100,000 / dish, and the co-culture time is 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h;

[0052] The concentrations of CPZ, Filipin III and EIPA used in the method are 28 μM, 2 μM and 50 μM respectively, and the co-culture time is 2 h.

[0053] In a specific embodiment, the AuHDs probe is applied to a method for cell internalization using dark field microscopy (DFM), comprising:

[0054] (1) Create a scattering curve:

[0055] The AuHDs probe was incubated with cells for different time periods (0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h), the quantity of AuHDs internalized by cells at different time periods was recorded, and then a curve showing the relationship between incubation time and internalization quantity was drawn.

[0056] (2) Determining the cellular endocytosis of the AuHDs under different inhibitors:

[0057] Different inhibitors were co-incubated with the cells for 2 h, and the content of AuHDs endocytosed by the cells was measured, so as to make a graph of the endocytic content of AuHDs under each group of inhibitors.

[0058] The present invention also provides a single particle tracking method based on colorimetric algorithm assistance using the gold homodimer AuHDs probe.

[0059] The present invention also provides a method for using the AuHDs probe to monitor the rotational motion of cargo vesicles in a cell transmembrane process under a dark field microscope (DFM), the method comprising:

[0060] (1) The AuHDs probe solution is co-cultured with cells to ensure that AuHDs can enter the cells.

[0061] (2) Counting the changes in the polar angle of the AuHDs probe across the cell membrane.

[0062] (3) Analyze the rotation dynamics of the AuHDs probe.

[0063] Wherein, the co-culture time with cells used in the method is 2h;

[0064] Among them, the number of statistical AuHDs particles used in the method is 28;

[0065] Among them, the statistical AuHDs polar angle changes used in the method are mainly concentrated in the transmembrane stage.

[0066] In a specific embodiment, the AuHDs probe is applied to a method for monitoring the rotational motion of cargo vesicles in a cellular process under a dark field microscope (DFM), the method comprising the following steps:

[0067] (1) Create a standard distribution image

[0068] The AuHDs probe is internalized into cells, and the polar angle changes of the probe during the transmembrane stage are recorded in real time through single particle tracking, and then a polar angle distribution image is made.

[0069] (2) Analysis of the rotational dynamics during the internalization of the AuHDs probe

[0070] The polar angle changes of the AuHDs probe during the cell internalization process were compared with the average polar angle of the AuHDs probe during the encapsulation stage to determine the rotational dynamic analysis of its unidirectional or bidirectional movement.

[0071] The beneficial effects of the present invention include: the gold homodimer AuHDs probe based on anisotropic scattering properties and uniform surface chemical properties proposed by the present invention can be obtained by a stepwise growth method and a DNA modification and purification synthesis method, and has uniform physical and chemical properties and dumbbell-shaped anisotropic scattering properties. Compared with the existing commonly used AuNRs and other probes, the gold homodimer AuHDs probe of the present invention can well avoid the toxicity of surfactants to cells and affect their endocytic activity; the gold homodimer AuHDs also has a high yield and stability, and can be applied to various complex biological environment; the gold homodimer AuHDs probe can also maintain its same composition, crystal structure and interface composition on the surface of the original gold seed, resulting in a clear unification of the surface chemical properties of the gold homodimer AuHDs probe; the above characteristics will also lead to different chemical and crystal properties of the gold homodimer AuHDs probe at the tip and side, thereby bringing about non-specific rotation of the gold homodimer AuHDs probe, so that the gold homodimer AuHDs probe forms an amorphous affinity with the surrounding membrane proteins, thereby reducing the unexpected movement of the gold homodimer AuHDs probe during endocytosis of the vesicle.

[0072] The present invention also discloses a single particle tracking (SPT) method assisted by a colorimetric algorithm (CBA). By using DFM and CBA-assisted single nanoparticle tracking technology, it can be used to accurately track the rotational motion of the AuHDs probe under a dark field microscope (DFM), and study the dynamic endocytosis process of AuHDs at the single particle level in a high-throughput manner. According to the rotational dynamics of AuHDs during the endocytosis process, its movement mode is determined. The AuHDs probe of the present invention has the ability to accurately study the dynamic process of cargo vesicles. The present invention paves the way for a better understanding of the physiological role of vesicle movement in receptor-mediated endocytosis. The present invention can be used to accurately track the rotational motion of cargo vesicles during endocytosis under a dark field microscope (DFM), thereby analyzing the dynamic process of cargo vesicles during vesicle movement. The present invention brings new inspiration to the development of anisotropic imaging probes in various cellular processes and the dynamic study of vesicle movement during endocytosis, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 :(a) Synthesis route of AuHDs probe; (b) Gel electrophoresis of AuHDs; (c) Hydration kinetic diameters of AuHDs and AuNPs; (d) TEM image of AuHDs; (e) Unification of size differences of AuHDs.

[0074] Figure 2 :(a) Stability test of AuHDs and AuNRs probes; (b) ) Yield analysis of AuHDs probes.

[0075] Figure 3 :(a) Color imaging of AuHDs under dark field microscope; (b) Relationship curve between polar angle and f(RG) of AuHDs in PBS, cMEM and simulation conditions; (c) Color transition process of a single AuHD in the polar angle range of 0-90° under polarization mode.

[0076] Figure 4 : (a) Scattering spectrum of AuHDs under dark-field microscopy and (b) UV-visible absorption spectrum.

[0077] Figure 5 : (a) Representative DFM images of AuHDs (left) and corresponding CBA-annotated images (right); (b) Uptake efficiency of AuHDs incubated with HeLa cells; (c) DFM images and (d) uptake efficiency of AuHDs under different pretreatment conditions: chlorpromazine (CPZ), filipin III, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and no treatment (control).

[0078] Figure 6 :(a) Time series of polarization angles of AuHDs, where the polarization angle is larger than the central polar angle φ of the package w (φ w +3SD) are shown in red, and the polarization angle is smaller than the central polar angle of the package (φ w (b) Color-coded diagram of the polar angle time series of 28 AuHDs independent particles. Red represents clockwise twisting motion, blue represents counterclockwise twisting motion, and gray represents the central polar angle of the wrapping. w .

[0079] Figure 7 :Schematic diagram of the synthesis of AuHDs and their dynamic analysis under dark-field microscopy. DETAILED DESCRIPTION

[0080] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0081] Example 1: Preparation and characterization of AuHDs probe

[0082] AuHDs were designed using a step-growth synthesis technique. 30 nm spherical AuNPs were used as growth seeds, and a mixture of Na3Ci and HAuCl4 was added to form new AuNPs on the surface of the growth seeds ( Figure 1a). Specifically, the reduction of HAuCl4 with Na3Ci leads to the formation of small gold nanoparticles in solution, and the newly formed nanoparticles randomly attach to the surface of existing nanoparticle seeds. The attached small nanoparticles then grow by attaching to the existing large nanoparticles through an “intra-particle maturation” process, resulting in the formation of dumbbell-shaped dimers between the two attached nanoparticles. More importantly, adjusting the Au content in the solution 3+ 、Au 0 The size of the nanoparticles formed on the seed surface was controlled by adjusting the ratio of the mixture of NH and NaOH, so that dimeric nanoparticles of the same size could be synthesized. Subsequently, in order to prevent the nanoparticles from aggregating in a complex living environment, the synthesized dimeric nanoparticles were modified with thiol DNA through the interaction of SH-Au.

[0083] Subsequently, these DNA-modified gold homopolymer nanoparticles (AuHDs) were purified by agarose gel electrophoresis and recovered by gel cutting ( Figure 1 b). Then, the hydrodynamic diameter of the measured particles was determined using a nanoparticle size analyzer of the Zetasizer Nano series (Malvern ZetasizerNano-ZS). The measured AuNPs and AuHDs samples were diluted to 0.1nM (1mL) with mili-Q water, and then the samples were placed in a special conventional cuvette and placed in a sample cell for particle size testing. The test results showed that the diameters of AuNPs and AuHDs were 36.9±0.6nm and 77.9±0.6nm, respectively ( Figure 1 c). AuHDs were characterized by TEM, and the TEM results showed that the sample yield was high ( Figure 1 d).

[0084] Finally, the uniformity of the dumbbell-shaped AuHDs was also evaluated by calculating the diameter difference (δ) at both ends of the AuHDs. The statistical results showed that δ was estimated to be centered at 1.6 ± 0.1 nm ( Figure 1 e), thus demonstrating the uniformity of the AuHDs surface.

[0085] Example 2: Stability and productivity of AuHDs probes.

[0086] First, the same concentration of AuHDs probe and control group AuNRs were centrifuged multiple times at the same speed (10000rpm, 10min), and then the color changes of AuHDs probe and control group AuNRs were observed to obtain the stability difference between AuHDs probe and control group AuNRs. The test results showed that compared with the control group AuNRs, the color of AuHDs probe remained unchanged even after multiple centrifugation, thus proving its good stability. (2) and (4) represent the color changes of AuNRs and AuHDs after multiple centrifugation, respectively, while (1) and (3) represent the colors of AuNRs and AuHDs without centrifugation in the initial stage.

[0087] Subsequently, the TEM data of the AuHDs probe in Example 1 was statistically analyzed by ImageJ software, and the data of 300 particles were analyzed to obtain the yield distribution of the AuHDs probe. The results showed that the yield of the AuHDs probe was as high as 80%, which exceeded most of the anisotropic imaging probes currently used in cell processes.

[0088] Example 3: Imaging mode of AuHDs probe under dark field microscope.

[0089] First, the AuHDs probe solution was dropped onto a silanized glass slide for 30 minutes, and then imaged and analyzed using a dark-field microscope. It was found that the representative AuHDs showed a large number of bright orange-green dots and a portion of green and red dots under a dark-field microscope ( Figure 3 a), which also explains that the imaging polar angles of the dimer on the slide are different, so that their imaging colors are also different.

[0090] Subsequently, polarization DFM was used to detect the color transition of a single AuHD immobilized on a glass surface (in a PBS solution environment). After successively rotating the polarizer by 3° relative to the analyzer, the color of the AuHD was observed to change from green to yellow, then from yellow to red, and finally from red to green ( Figure 3 c). Finally, we tested whether the complex biological environment affects the ability of AuHDs to determine their polar angles, which is crucial for observing the rotational dynamics of anisotropic probe vesicles in vivo. The results showed that after AuHD was incubated in cMEM (containing 10% fetal bovine serum) using polarized DFM, the color transition process of AuHD was still consistent with the color change of PBS. In addition, the present invention also uses a dual-channel differential equation to calculate the relationship between the polar angle (φ) of the particle and the intensity of RG based on the inherent characteristics of the dark field microscope, which satisfies the following equation: f(RG) = (1.36sin 2 φ-0.36sin 4φ). The intensity and polar angle changes of AuHD RG in PBS and cMEM also satisfy the curve equation ( Figure 3 b), which also shows that the scattering properties of AuHDs can satisfy the monitoring of their motion patterns under DFM.

[0091] Example 4: Spectral characteristics of AuHDs probe under dark field microscope.

[0092] First, the AuHDs probe solution was dropped on a silanized glass slide for 30 minutes, and then the spectrum was analyzed by dark field microscopy. Due to its dumbbell-shaped structure, the synthesized AuHDs exhibited two different oscillation modes under incident light, namely the longitudinal surface plasmon resonance (LSPR) along its long axis and the transverse LSPR along its short axis. Its corresponding scattering spectrum under DFM showed a peak at 565nm, corresponding to the transverse vibration mode, and another peak at 650nm, corresponding to the longitudinal vibration mode ( Figure 4 a). In addition, the intensity of the longitudinal scattered emission slightly increases compared to the transverse scattered emission. This result indicates that the dumbbell-shaped structure of the synthesized AuHDs leads to anisotropic optical properties of the longitudinal and transverse LSPRs.

[0093] Subsequently, the optical absorption was measured by UV-visible spectroscopy. The AuHDs of the present invention exhibited two peaks in the UV-visible spectrum, one centered at 524 nm and the other at 634 nm ( Figure 4 b), corresponding to the combination of the transverse oscillation mode and the longitudinal oscillation mode, this result also proves that AuHDs have anisotropic scattering characteristics of the longitudinal and transverse LSPR resonance frequencies.

[0094] Example 5: Cell internalization process of AuHDs probe under dark field microscope

[0095] AuHDs were incubated with cells for different time periods (0.5h, 1h, 2h, 4h, 8h, 12h). After incubation, the cells were washed three times with PBS to remove the particles that were not internalized. Then, fresh MEM medium was added for dark field microscopy. The exposure time for the shooting was 60ms, and the number of cells counted was 20. CBA was used to obtain the number of AuHDs internalized by cells at different time stages ( Figure 5 a). DFM images showed that a large number of bright yellow dots representing AuHDs were scattered throughout the cells, and CBA analysis showed that the number of AuHDs in the cells increased over time ( Figure 5b), these results indicate that AuHDs indeed entered the cells. Subsequently, the present invention further studied the internalization mechanism of AuHDs, using various endocytosis inhibitors, chlorpromazine (CPZ), filipin III, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and no treatment (2h) as a control ( Figure 5 c), after pretreatment of cells with Filipin III, CPZ and EIPA, the absorption efficiency of cells decreased by 36%, 47% and 49%, respectively ( Figure 5 d) This result indicates that AuHDs enter cells through multiple pathways, including caveolin- and lectin-mediated endocytosis and macrophage action, confirming that the AuHDs of the present invention are transported into cells by being encapsulated in endocytic vesicles.

[0096] Example 6: AuHDs probe was used to monitor the rotational motion of cargo vesicles in cellular processes under dark-field microscopy.

[0097] The AuHDs probe was incubated with cells for 2 h to ensure that the transmembrane movement of AuHDs could be observed. The rotational movement of AuHDs during transmembrane movement can be tracked and acquired by CBA-assisted single particle tracking (SPT). The present invention further analyzed the polar angle changes of AuHDs in 28 cargo vesicles ( Figure 6 b) According to the polar angle over time, φ W Based on the changes in the content of the vesicles, the present invention observed that the cargo vesicles exhibited two different reversible rotation dynamics before the vesicle movement: clockwise twisting, polar angle increase and release; counterclockwise twisting, polar angle decrease and release ( Figure 6 a)). Interestingly, from the rotational dynamics of 28 cargo vesicles, it was found that 4 cargo vesicles rotated in a clockwise direction and 18 rotated in a counterclockwise direction ( Figure 6 b), and the rest are bidirectional motion modes. Considering that the intermittent twisting of each cargo vesicle is a clockwise or counterclockwise process, the present invention can conclude that the vesicle twisting driven by the nanomechanical scaffold is mainly unidirectional.

[0098] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for preparing a gold homodimer AuHDs probe, characterized in that: The method comprises selecting 30-32 nm gold nanoparticles AuNPs as gold seeds, adding a mixture of Na3Ci and HAuCl4, and attaching and growing on the gold seeds through an "intra-particle maturation" process, thereby forming a dumbbell-shaped dimer between two attached nanoparticles; The method comprises the following steps: (1) synthesizing gold nanoparticles with a size of 30-32 nm and using them as a seed solution for the reaction; (2) on the basis of step (1), sodium hydroxide NaOH, sodium citrate Na3Ci, and tetrachloroauric acid trihydrate HAuCl43H2O are added in sequence to react and obtain gold nano dimers; (3) On the basis of step (2), SH-DNA is added to prevent particle aggregation by utilizing the strong interaction of Au-S bonds; (4) Based on the step (3), the desired dimer band is separated by agarose gel electrophoresis, and the gold homodimer AuHDs probe is obtained by gel cutting and recovery.

2. The preparation method according to claim 1, characterized in that In the step (1), HAuCl4 and Na3Ci are added at the initial stage to synthesize the precursor solution required for the reaction; wherein the reaction temperature is 100-140° C. and the reaction time is 40-60 min; and / or, In the step (1), HAuCl4 is continuously added to the precursor solution to synthesize AuNPs with a size of 30 nm; wherein the reaction temperature is 90-100° C. and the reaction time is 1-2 h; and / or, In the step (2), NaOH and NaCi3 are added to the AuNPs solution synthesized in the step (1) in sequence, the reaction temperature is 15-25°C, and the reaction time is 10-20 min; HAuCl4 is added to the above solution, the reaction temperature is 30-37°C, and the reaction time is 30-45 min; the volume ratio of the NaOH, Na3Ci, HAuCl43H2O and gold nanoparticles is 20-30 μL: 30-50 μL: 4-8 μL: 4-8 μL; the concentrations of the NaOH, Na3Ci, HAuCl43H2O and gold nanoparticles are all 25 mM.

3. The preparation method according to claim 2, characterized in that: In the step (3), SH-DNA is added; the reaction temperature is -60 to -80°C, and the reaction time is 5 to 10 minutes; and / or, In the step (4), agarose gel electrophoresis technology is used to cut the gel to recover the desired product bands to increase the yield of the gold homodimer AuHDs sample; the reaction voltage is 100 V, the reaction time is 30 min, and the buffer used is 0.5×tris-borate-EDTA.

4. The gold homodimer AuHDs probe prepared by the method according to any one of claims 1 to 3; characterized in that: The probe has uniform physicochemical properties and anisotropic scattering characteristics in a dumbbell shape.

5. The use of the gold homodimer AuHDs probe as described in claim 4 in the imaging mode under dark field microscopy, in the spectral characteristics under dark field microscopy DFM and UV-visible light, in the precise tracking of the cell endocytic movement under dark field microscopy DFM, and in the precise tracking of the rotational movement of cargo vesicles during endocytosis under dark field microscopy DFM.

6. A method for applying the gold homodimer AuHDs probe as claimed in claim 4 in an imaging mode under a dark field microscope DFM, characterized in that: The method comprises: (1) titrating the gold homodimer AuHDs probe solution on a silanized glass slide, and performing imaging detection using a dark field microscope; (2) The R and G scattering intensities of the gold homodimer AuHDs probe under dark field were measured by CBA algorithm, and the polar angle (φ) of the gold homodimer AuHDs probe was calculated according to the dual-channel differential equation: f(RG) = (1.36sin 2 φ-0.36sin 4 φ).

7. A method for applying the gold homodimer AuHDs probe as claimed in claim 4 to spectral characteristics under dark field microscopy DFM and UV-visible spectroscopy, characterized in that: The method comprises the steps of: (1) titrating the gold homodimer AuHDs probe solution on a silanized glass slide, and performing optical detection under a dark field microscope and UV-visible spectroscopy; (2) The gold homodimer AuHDs probe is subjected to spectral analysis by scattering and ultraviolet spectrometer.

8. A method for accurately tracking the cellular endocytosis process under dark field microscopy (DFM) using the gold homodimer AuHDs probe as claimed in claim 4, characterized in that: The method comprises: (1) co-culturing the gold homodimer AuHDs probe with cells to allow the gold homodimer AuHDs probe to enter the cells; (2) The cellular interactions of the gold homodimer AuHDs probe during cellular endocytosis were analyzed by using various endocytosis inhibitors, including chlorpromazine (CPZ), filipin III, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), and it was determined that the gold homodimer AuHDs probe can be used to monitor the movement of cargo vesicles during endocytosis.

9. A single particle tracking method based on colorimetric algorithm assistance, characterized in that: A method for accurately tracking the rotational motion of cargo vesicles during endocytosis under a dark field microscope (DFM) using the gold homodimer AuHDs probe as claimed in claim 4, the method comprising: (1) co-culturing the gold homodimer AuHDs probe with cells to allow the gold homodimer AuHDs probe to enter the cells; (2) The rotational motion information of the gold homodimer AuHDs probe was extracted by calculating its polar angle from the color dark-field image using CBA-assisted TrackPy and a two-channel differential equation, thereby accurately monitoring the dynamics of cargo vesicles during vesicle movement.

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