Dual-mode nanoprobe as well as preparation method and application thereof

Abstract

The invention relates to a dual-mode nanoprobe as well as a preparation method and application thereof, and belongs to the technical field of nanoprobes. The dual-mode nanoprobe comprises gold-core and silver-shell nanoparticles, and a protective agent and a polypeptide chain which are respectively modified on the surfaces of the gold-core and silver-shell nanoparticles, the polypeptide chain comprises a mitochondrial targeting peptide, and a sulfydryl group and a fluorescent molecule which are respectively modified at two ends of the mitochondrial targeting peptide; the gold-core silver-shell nanoparticles and the polypeptide chain are connected through a silver-sulfur covalent bond; and the sequence of the mitochondrial targeting peptide is MLALLGWWWFFSRKKC. The dual-mode nanoprobe not only has excellent selectivity and sensitivity to mitochondrial ROS, but also can monitor the dynamic change of the mitochondrial ROS in a cell apoptosis process in real time, is helpful to deeply understand the effect of the ROS in a mitochondrial mediated cell apoptosis signal channel, and is helpful to deeply understand a signal transduction mechanism in the cell apoptosis process.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoprobes, and in particular relates to a dual-mode nanoprobe and a preparation method and application thereof. Background Art

[0002] Fumonisin B1 (Fumonisin FB1) is a hydrophilic metabolite produced by Fusarium moniliforme, a Fusarium fungus. FB1 mainly contaminates food, cereals and their products. Long-term exposure to this type of mycotoxin will pose a threat to the health of exposed individuals. The toxic mechanism of FB1 is to interfere with the normal pathway of de novo biosynthesis of sphingolipids by acting as a competitive inhibitor of ceramide synthase. At the same time, the toxic products of sphingolipid metabolism disorders can cause oxidative stress and cell apoptosis. Studies have shown that FB1 toxin can induce cellular oxidative stress, leading to cell apoptosis and fibrosis. Among them, inducing cell apoptosis is one of the important ways for FB1 to exert its pathogenic effects and produce immunotoxicity, and it is also a hot issue in the field of mycotoxin research in recent years.

[0003] Reactive oxygen species (ROS) are considered to be an important indicator of cell apoptosis. Under normal physiological conditions, the intracellular oxidation / antioxidation system is in a state of balance, and the intracellular ROS content is low, which will not cause damage to the cells. However, when tissue cells are damaged, mitochondrial respiratory chain complexes I and III will leak a large amount of electrons, prompting the production of a large amount of ROS, breaking the intracellular oxidation / antioxidation balance, and then causing mitochondrial dysfunction, leading to mitochondrial autophagy and cell apoptosis. Mitochondria are one of the most important components of cells. They are both the main site of ROS production and a sensitive target of their action. There is a close relationship between ROS and mitochondrial damage. Therefore, accurate detection of ROS in mitochondria is extremely critical for in-depth exploration of the signal transduction mechanism in the process of cell apoptosis.

[0004] Commonly used ROS detection techniques include colorimetry, electrochemical methods, and fluorescence imaging. However, the first two methods are complicated to operate, usually require the destruction of tissues or cells, and cannot detect ROS levels in living cells. Although fluorescence imaging has high sensitivity and selectivity, fluorescent probes have problems such as photobleaching, which limits their stability and reliability for long-term or dynamic detection. To overcome these challenges, nanotechnology biosensors based on different mechanisms have been developed to detect intracellular ROS. Such as nanospectral detection, nanofluorescent probes, and electrochemical biosensors. However, the intracellular environment is complex, and single-mode detection technology is prone to false positive interference problems. In addition, real-time in situ monitoring and dynamic tracking of ROS changes are extremely important for a deep understanding of the mechanism of cell apoptosis.

[0005] Surface enhanced Raman scattering (SERS) is a highly sensitive spectroscopy technique that can significantly enhance the Raman signals of molecules on the surface of nanomaterials. The detection advantage of SERS lies in its high sensitivity and high selectivity, and it can detect target molecules at extremely low concentrations. Polypeptide chains can achieve efficient recognition and binding of target molecules through specific sequences, thereby improving the selectivity of detection, but single-mode detection technology is susceptible to background interference and may produce false positive signals. Therefore, the development of a dual-mode nanoprobe that combines multiple detection mechanisms has important research significance and application prospects for improving the reliability and sensitivity of detection. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a dual-mode nanoprobe and a preparation method and application thereof. Gold core and silver shell nanoparticles Au@AgNPs are used as a substrate, and a protective agent and a polypeptide chain are modified on the surface of Au@AgNPs to obtain a dual-mode nanoprobe (Au@AgNPs-MLS), which realizes the precise positioning of the dual-mode nanoprobe in cell mitochondria and the detection of subcellular structural biological molecules.

[0007] The first object of the present invention is to provide a dual-mode nanoprobe, which includes gold core-silver shell nanoparticles, and a protective agent and a polypeptide chain respectively modified on the surface of the gold core-silver shell nanoparticles; the polypeptide chain includes a mitochondrial targeting peptide, and a thiol group and a fluorescent molecule respectively modified at both ends of the mitochondrial targeting peptide; the gold core-silver shell nanoparticles and the polypeptide chain are connected by a silver-sulfur covalent bond; the sequence of the mitochondrial targeting peptide is MLALLGWWWFFSRKKC.

[0008] In one embodiment of the present invention, the particle size of the gold core-silver shell nanoparticles is 18nm-24nm; the thickness of the silver layer is 4nm-5nm.

[0009] In one embodiment of the present invention, the protective agent is selected from methoxy polyethylene glycol thiol and / or Tween-20.

[0010] In one embodiment of the present invention, the molar concentration ratio of the gold core-silver shell nanoparticles, the protective agent and the polypeptide chain is 1:(2500-5000):(1200-1600).

[0011] In one embodiment of the present invention, the fluorescent molecule is selected from one or more of rhodamine B, fluorescein isothiocyanate, carboxyfluorescein and anthocyanin fluorescent dyes.

[0012] The second object of the present invention is to provide a method for preparing the dual-mode nanoprobe, comprising the following steps: sequentially adding a protective agent solution and a polypeptide chain solution to a gold core-silver shell nanoparticle solution for incubation to obtain the dual-mode nanoprobe.

[0013] In one embodiment of the present invention, the concentration of the gold core-silver shell nanoparticle solution is 2 nM; the concentration of the protective agent solution is 0.1 mM-0.2 mM; and the concentration of the polypeptide chain solution is 240 μM-320 μM.

[0014] In one embodiment of the present invention, the volume ratio of the gold core-silver shell nanoparticle solution, the protective agent solution, and the polypeptide chain solution is 1000:(50-100):(10-20).

[0015] The third object of the present invention is to provide an application of the dual-mode nanoprobe in ROS detection.

[0016] In one embodiment of the present invention, the concentration of the dual-mode nanoprobe in the application is 0.25nM-1nM.

[0017] In one embodiment of the present invention, the application is based on the "on-off" conversion of surface enhanced Raman scattering (SERS) and fluorescence resonance energy transfer (FRET). A polypeptide chain (mitochondrial targeting peptide containing fluorescent molecules and thiol groups) is modified on the surface of the gold core silver shell nanoparticles, which can effectively target the mitochondrial site of the cell. When the silver shell is intact, the fluorescence signal is quenched and the Raman signal is enhanced. When ROS is present, it will react with the silver surface, resulting in oxidation and dissolution of silver, and this reaction will destroy the silver shell structure. When the silver shell is etched by ROS, the polypeptide chain falls off, the fluorescent molecules are away from the surface of the material, the Raman signal is weakened, and the fluorescence signal is restored. The etching effect of ROS on the silver layer causes the SERS in the system to be "turned off" and the fluorescence to be "turned on". Based on this principle, the dual-mode nanoprobe that is easily absorbed by cells can realize real-time dynamic detection and in situ imaging of mitochondrial ROS in living cells.

[0018] The technical solution of the present invention has the following advantages over the prior art:

[0019] (1) The dual-mode nanoprobe described in the present invention combines the dual advantages of Raman and fluorescence. The silver layer on the surface of the gold core silver shell nanoparticles can produce a very high electric field effect and has a good surface enhanced Raman effect. After the dual-mode nanoprobe enters the cell, the initial fluorescence signal is quenched and the fluorescence background signal is very weak. As cell apoptosis proceeds, the fluorescence signal gradually recovers and the Raman signal gradually decreases. Through Raman and fluorescence dual imaging, the changes in mitochondrial ROS can be more intuitively reflected and monitored, thereby realizing real-time dynamic detection and in situ imaging of mitochondrial ROS during cell apoptosis. By combining SERS and fluorescence technology, the probe can effectively eliminate the interference of the intracellular environment and provide more reliable detection results.

[0020] (2) The dual-mode nanoprobe described in the present invention not only has excellent selectivity and sensitivity to mitochondrial ROS, but also can monitor the dynamic changes of mitochondrial ROS in real time during cell apoptosis, which helps to gain a deeper understanding of the role of ROS in the mitochondria-mediated apoptosis signaling pathway, and is conducive to a deeper understanding of the signal transduction mechanism in the process of cell apoptosis, and effectively solves the false positive interference problem that may occur in single-mode detection technology.

[0021] (3) The dual-mode nanoprobe of the present invention modifies the gold core-silver shell nanoparticles with a protective agent and a polypeptide chain, so that the gold core-silver shell nanoparticles are wrapped on the surface, preventing the contact and aggregation of the gold core-silver shell nanoparticles, and improving the stability of the dual-mode nanoprobe. At the same time, the modification of the polypeptide chain can effectively target the mitochondrial part of the cell to detect biological molecules in the subcellular structure; the modification of the protective agent can stabilize the probe structure, thereby improving the stability of the dual-mode nanoprobe.

[0022] (4) The dual-mode nanoprobe described in the present invention is easy to prepare, has low toxicity and no damage to cells, is easily absorbed by cells, and has good biocompatibility.

[0023] (5) The application described in the present invention utilizes ROS to etch the silver layer on the surface of the dual-mode nanoprobe, causing the surface-modified targeting peptide carrying fluorescent molecules to fall off, thereby achieving fluorescence signal recovery and Raman signal attenuation, and designing a highly sensitive Raman-fluorescence dual-mode nanoprobe for the detection of mitochondrial ROS. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 The characterization results of Au@AgNPs in Test Example 1 of the present invention; wherein A is an electron microscope image, B is an average particle size distribution, and C is an ultraviolet-visible absorption spectrum;

[0026] Figure 2 The fluorescence results of the dual-mode nanoprobe after incubation with polypeptide chain solutions of different concentrations in Test Example 2 of the present invention;

[0027] Figure 3 The Raman results of the dual-mode nanoprobe after incubation with polypeptide chain solutions of different concentrations in Test Example 2 of the present invention;

[0028] Figure 4 The stability test results of the dual-mode nanoprobe in Test Example 3 of the present invention;

[0029] Figure 5 The reproducibility experimental results of the dual-mode nanoprobe in Test Example 4 of the present invention;

[0030] Figure 6 This is the ultraviolet absorption spectrum of the dual-mode nanoprobe used for in vitro detection of H2O2 in Test Example 5 of the present invention;

[0031] Figure 7 The fluorescence (A) and Raman (B) detection results of the dual-mode nanoprobe used for in vitro detection of H2O2 in Test Example 5 of the present invention;

[0032] Figure 8 The cytotoxicity results of different concentrations of FB1 toxin in test example 6 of the present invention when incubated with HepG2 cells for 12 hours;

[0033] Fig. 9 The cytotoxicity results of the dual-mode nanoprobes of different concentrations in Test Example 7 of the present invention when incubated with HepG2 cells for different times;

[0034] Fig.10 is the uptake of the dual-mode nanoprobe by HepG2 cells (AB) and mitochondria (C) in Test Example 8 of the present invention;

[0035] Fig.11 The detection of the content of ROS in the apoptosis process of living cells by the commercial fluorescent probe in Test Example 9 of the present invention;

[0036] Fig.12 The dual-mode nanoprobe in Test Example 9 of the present invention is used for laser confocal imaging of mitochondrial ROS in living cells;

[0037] Fig.13 Comparison of the imaging results of the dual-mode nanoprobe and the commercial fluorescent probe in Test Example 9 of the present invention;

[0038] Fig.14 The dual-mode nanoprobe in Test Example 10 of the present invention is used for SERS imaging of mitochondrial ROS in living cells;

[0039] Fig.15 The dual-mode nanoprobe in Test Example 10 of the present invention is used for SERS spectroscopy detection of mitochondrial ROS in living cells. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] In the present invention, unless otherwise explained, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0042] In the present invention, unless otherwise stated, the term "and / or" used in the present invention includes any and all combinations of one or more of the associated listed items.

[0043] In the present invention, unless otherwise stated, the experimental methods used in the embodiments of the present invention are conventional methods unless otherwise stated, and the materials, reagents, etc. used are all commercially available unless otherwise stated.

[0044] In the present invention, unless otherwise specified, the polypeptide chain used in the embodiments of the present invention was purchased from Sangon Biotechnology (Shanghai) Co., Ltd. The polypeptide chain is a mitochondrial targeting peptide modified with rhodamine B at the N-terminus and thiol at the C-terminus, and the sequence of the mitochondrial targeting peptide is NC: MLALLGWWWFFSRKKC.

[0045] Example 1

[0046] The dual-mode nanoprobe and the preparation method thereof of this embodiment specifically include the following steps:

[0047] S1. Preparation of gold core-silver shell nanoparticles (Au@AgNPs): 100 mL of HAuCl4 aqueous solution (1 mM) was heated to boiling under vigorous stirring, and 15 mL of sodium citrate solution (1%) was quickly added. After continuous stirring and heating for 10 min, it was cooled to room temperature to obtain an AuNPs solution with a particle size of 13 nm; 6 mL of AuNPs solution was mixed with 800 μL of ascorbic acid solution (100 mM), and then 1 mL of AgNO3 solution (1.0 mM) was added dropwise. After stirring for 1 h, it was centrifuged at 10000 rpm for 10 min, and then resuspended in 0.5% PVP solution and fixed to 2 nM to obtain Au@AgNPs solution;

[0048] S2. Preparation of dual-mode nanoprobe (Au@AgNPs-MLS): Add 50 μL of mPEG-SH solution (0.1 mM) to 1 mL of Au@AgNPs solution and incubate for 2 h. After centrifugation and washing, add 10 μL of polypeptide chain solution (MLS solution, 280 μM) and continue incubating for 12 h (200 rpm, 37 °C). Centrifuge to remove excess polypeptide chains and resuspend to the original volume to obtain Au@AgNPs-MLS solution.

[0049] Test Case 1 Characterization

[0050] Based on Example 1, Au@AgNPs were characterized and the results were as follows Figure 1 As shown. Figure 1 A shows that the particle size of Au@AgNPs is uniform, and the morphology and dispersion are good; Figure 1 B shows that the average particle size of Au@AgNPs is about 22nm; Figure 1C It can be seen that the UV absorption spectrum of Au@AgNPs is between 405nm-520nm, which partially overlaps with the emission spectrum of Rb, indicating that Au@AgNPs can quench the fluorescence of Rb.

[0051] Test Example 2: Stability Experiment

[0052] Based on Example 1, the optimal loading amount of the dual-mode nanoprobe with different concentrations of polypeptide chain solution was explored: 10 μL of polypeptide chain solution with different concentrations was incubated with 1 mL of nanoprobe for 12 h (200 rpm, 37°C) to make the final concentrations of 40 μM, 80 μM, 120 μM, 200 μM, 240 μM, 280 μM, and 320 μM. The decrease in the fluorescence signal of the supernatant after centrifugation and the enhancement of the Raman signal of the dual-mode nanoprobe were used to prove that the nanomaterial was modified by the polypeptide chain. The results are as follows: Figure 2-Figure 3 As shown. Figure 2 It can be seen that with the increase of polypeptide chain concentration, the difference in fluorescence intensity of the solution supernatant before and after hybridization gradually increases; when the concentration reaches 2.8μM, it remains almost unchanged. Figure 3 It can be seen that with the increase of peptide concentration, the Rb group at 1650cm -1 The SERS intensity of the characteristic peak at the concentration increased significantly; when the concentration reached 280 μM, the intensity of the characteristic peak remained roughly unchanged.

[0053] Test Example 3 Stability experiment in solvent

[0054] (1) Characterization of the salt stability of the dual-mode nanoprobe by UV-visible spectroscopy: Au@AgNPs-MLS was resuspended in a NaCl buffer solution containing different concentrations (0 mM, 200 mM, 300 mM, 400 mM, 500 mM) for 30 min, and then UV-visible spectroscopy analysis was performed. The results are shown in Figure 2. Figure 4 As shown in A.

[0055] (2) Characterization of pH stability of dual-mode nanoprobe by UV-visible spectroscopy: Au@AgNPs-MLS was resuspended in potassium dihydrogen phosphate-sodium dihydrogen phosphate buffer solutions with different pH values ​​(pH = 5, 6, 7, 8, 9) for 2 h, and then analyzed by UV-visible spectroscopy. The results are shown in Figure 2. Figure 4 As shown in B.

[0056] (3) Raman experiment characterization of the solvent stability of the dual-mode nanoprobe: Au@AgNPs-MLS was resuspended in PBS buffer solution (0.01 M, pH 7.4) and DMEM basic culture medium and incubated for 0 h, 8 h, 16 h and 24 h, and the changes in the SERS signal of the Rb group were detected. The results are shown in Figure 2. Figure 4 As shown in C.

[0057] from Figure 4 A- Figure 4 B shows that the UV absorption spectrum of Au@AgNPs-MLS does not change significantly under different concentrations and pH conditions; this is because the mPEG-SH and polypeptide chains modified on the surface of Au@AgNPs-MLS prevent the aggregation of Au@AgNPs, making Au@AgNPs-MLS have excellent anti-interference ability and stability. Figure 4 C It can be seen that after Au@AgNPs-MLS is resuspended in different solvents for a period of time, its Raman signal remains stable, which shows that the influence of culture medium and PBS buffer on Au@AgNPs-MLS is negligible.

[0058] Test Example 4: Signal reproducibility experiment

[0059] The Au@AgNPs-MLS solution was dispersed on a glass slide wrapped with tin foil and dried for SERS measurement with a Raman excitation wavelength of 633 nm and a scanning range of 200 cm -1 Up to 2000cm -1 The test was repeated 3 times to obtain the average intensity of fluorescence and SERS, and the Raman spectra of Au@AgNPs-MLS at 20 different points were collected to evaluate its signal reproducibility. The results are shown in Figure 5 As shown. Figure 5 It can be seen that the SERS peak intensities at 20 different points are basically the same, and the relative standard deviation is 4.510%, which indicates that the prepared Au@AgNPs-MLS has excellent Raman signal reproducibility.

[0060] Test Example 5 Dual-mode nanoprobe for in vitro H2O2 detection

[0061] (1) During the silver etching reaction, 10 μL of 10 μM H2O2 solution was added to 100 μL Au@AgNPs-MLS solution (2 nM) and the reaction was carried out at room temperature for different time periods. The changes in the UV-visible spectrum were observed. The results are shown in Figure 2. Figure 6 shown.

[0062] (2) 10 μL of H2O2 solutions of different concentrations (0-10 μM) were added to 100 μL of Au@AgNPs-MLS solution (2 nM) and reacted at room temperature for 6 h. After centrifugation, the supernatant was collected, and 540 nm was used as the excitation wavelength, and the fluorescence signal was collected at 590 nm. The precipitate was dispersed on a glass slide wrapped in tin foil and dried for SERS measurement. The Raman excitation wavelength was 633 nm and the scanning range was 200 cm -1 Up to 2000cm -1 The test was repeated 3 times to obtain the average intensity of fluorescence and SERS. Figure 7shown.

[0063] from Figure 6 It can be seen that the position of the UV-visible spectrum shifts, and its UV peak gradually moves to the right from 405nm, and finally remains unchanged at 530nm, indicating that the etching of the silver layer is completed. Figure 7 It can be seen that as the concentration of H2O2 increases, the collected fluorescence signal gradually increases and the Raman signal weakens. In the range of 0.2μM-2μM, a good linear relationship is observed between the fluorescence intensity of the Rb fluorescent molecule and the concentration of H2O2. The regression equation is y=1852.22354·X+592.81272(R 2 =0.99703), the detection limit (LOD) is 66.44nM. Similarly, the H2O2 concentration in the range of 0.2μM-2μM is similar to that at 1650cm -1 There is also a good linear relationship between the SERS intensities at . The regression equation is y = -5508.10562·X + 13311.6231 (R 2 =0.99573), and the detection limit (LOD) was 38.13 nM, indicating that Au@AgNPs-MLS has good sensitivity and in vitro response.

[0064] Test Example 6: CCK-8 method to measure the cytotoxicity of FB1 toxin

[0065] FB1 toxin was diluted to different concentrations (1 μM, 10 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM) in cell culture medium; cells were seeded into 96-well plates at a density of 5000 per well. After 24 h of incubation in a 37°C, 5% CO2 incubator, medium containing different concentrations of FB1 toxin was added to each well. After further incubation for 12 h, 10 μL of CCK-8 reagent was added to each well.

[0066] After incubation for 4 h, the absorbance was measured at 450 nm. The relative cell survival rate was calculated using the following formula: relative cell survival rate (RGR) = (absorbance value of the experimental group - CCK8 background) / (experimental value of the control group - CCK8 background) × 100%. Figure 8 As shown in A. Figure 8 As can be seen in A, when HepG2 cells were exposed to different concentrations of FB1 toxin for 12 h, cell viability decreased in a dose-dependent manner.

[0067] The IC50 of FB1 toxin on HepG2 cells was fitted by Origin software. The results are as follows Figure 8 As shown in B. Figure 8 As can be seen from B, the IC50 value is about 245 μM. This concentration is used as the standard and the working concentration of the toxin in subsequent experiments.

[0068] Test Example 7 CCK-F method to evaluate the cytotoxicity of dual-mode nanoprobes

[0069] HepG2 cells were incubated overnight in a 96 black ELISA plate until they adhered to the wall, and then different concentrations (0.25nM, 0.5nM, 0.75nM and 1nM) of Au@AgNPs-MLS solution were added. After continued incubation for different time periods (6h, 12h and 24h), the cells were washed several times with PBS buffer. 100μL CCK-F reagent was added and placed in a cell culture incubator to react in the dark for 30min, and then the fluorescence intensity was measured immediately. The excitation wavelength was 494nm and the emission wavelength was 517nm. The cell activity was proportional to the fluorescence intensity. When evaluating the cell viability, the cells without drug administration were used as the blank control group for calculation. The results are shown in Fig. 9 As shown. Fig. 9 It can be seen that HepG2 cells still showed more than 80% activity after incubation with high concentrations of nanoprobes for 24 h, indicating that Au@AgNPs-MLS has low toxicity and is suitable for the cell environment.

[0070] Test Example 8: Study on the cellular uptake and distribution of dual-mode nanoprobes

[0071] (1) HepG2 cells were cultured at 5×10 5 The cells were inoculated into 6-well plates at a density of 100 μg / mL. After the cells adhered to the wall and grew for 24 hours, the old culture medium was removed. Subsequently, 0.75 nM Au@AgNPs-MLS nanoprobe solution was added to each well and incubated for 0 h, 4 h, 6 h, 8 h, 12 h, and 14 h, respectively. The cells were then digested with trypsin, collected and counted after centrifugation, and finally an equal amount of aqua regia was added to digest overnight to break the cells. ICP-MS was used for analysis to calculate the number of Au@AgNPs-MLS that entered the cells. ICP-MS was used to quantitatively analyze the cellular uptake of Au@AgNPs-MLS and obtain the standard curve of Au@AgNPs-MLS and the corresponding CPS value ( Fig.10 A), the uptake of nanoprobes by HepG2 cells at different incubation times can be calculated, and the results are shown in 10B. Fig.10 B shows that the number of intracellular probes increases with the increase of incubation time until it reaches saturation around 12h-14h. 5 The concentration of nanoprobes in cells was about 52 pM.

[0072] After extracting mitochondria using a mitochondrial extraction kit, the same method was used to detect the content of Au@AgNPs-MLS in mitochondria. The results are as follows Fig.10 C. From Fig.10C shows that the absorption of Au@AgNPs-MLS by mitochondria is 32.2pM / 10 5 By comparing the response value after the whole cell endocytosed the probe, it was calculated that the Au@AgNPs-MLS taken up by mitochondria accounted for 61.9% of the total cellular endocytosis, which can prove that Au@AgNPs-MLS can effectively target mitochondria and achieve mitochondrial localization of the nanoprobe.

[0073] Test Example 9

[0074] (1) Determination of ROS content in living cell apoptosis using commercial fluorescent probes: HepG2 cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μM / mL in a glass-bottomed dish. After the cells adhered to the wall, 1 mL of DMEM medium containing 245 μM FB1 was added to the dish and incubated for different times (0 h, 2 h, 4 h, 6 h, 8 h and 12 h). After the co-incubation was completed, the medium containing FB1 was removed and replaced with 1 mL of DCFH-DA solution diluted with serum-free culture medium. Subsequently, the cells were placed in a cell culture incubator at 37 ° C for 20 min to ensure that DCFH-DA fully entered the cells and was converted into DCF, which reflects the level of reactive oxygen in the cells. The cells were washed to remove the fluorescent probes that did not enter the cells. The treated cells were directly observed using a laser confocal microscope to evaluate the changes in the level of reactive oxygen in HepG2 cells under FB1 stimulation for different times. The excitation light source was 488 nm. The imaging results are shown in Figure 2. Fig.11 As shown. Fig.11 It can be seen that as the intracellular ROS concentration increases, the fluorescence intensity also increases.

[0075] (2) Dual-mode nanoprobes for living cell fluorescence imaging: HepG2 cells were cultured at 1×10 5 The cells were inoculated into a glass-bottomed dish at a density of 100 μg / mL. After overnight incubation, the old culture medium was removed and Au@AgNPs-MLS was added to co-culture with the cells for 12 hours. The cells were then washed with PBS several times and then added with a culture medium containing 245 μM FB1 and incubated for different time periods (0 hours, 2 hours, 4 hours, 6 hours, 8 hours, and 12 hours). The supernatant was aspirated and washed with PBS several times. The cells were stained with DAPI for 5 minutes and fixed with 4% paraformaldehyde for 10 minutes. The laser confocal microscope was used as the excitation light source at 405 nm and 543 nm. Dual-channel independent imaging was performed. The results are shown in Figure 2. Fig.12 As shown. Fig.12 It can be seen that with the increase of FB1 toxin culture time, the cell apoptosis process deepens; the intracellular ROS level increases, the silver layer on the surface of Au@AgNPs-MLS is gradually etched, and the Rb group is released from the surface of the nanoprobe. The imaging results show that the quenched fluorescence gradually recovers.

[0076] The results of Au@AgNPs-MLS and DCFH-DA were compared. Fig.13 As shown. Fig.13 It can be seen that the fluorescence increase trends of Au@AgNPs-MLS and DCFH-DA are consistent, indicating that the dual-mode nanoprobe can effectively measure the changes of intracellular ROS and the data are reliable.

[0077] Test Example 10 Dual-mode nanoprobe for Raman detection and imaging in living cells

[0078] HepG2 cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / mL into a small glass-bottomed dish. After overnight incubation for adhesion, the old culture medium was removed and Au@AgNPs-MLS was added to co-culture with the cells for 12 hours. The cells were then washed with PBS several times and then cultured with 250 μM FB1. The culture medium was added and incubated for different times (0 h, 2 h, 4 h, 6 h, 8 h and 12 h). The supernatant was aspirated and washed several times with PBS and then fixed with 4% paraformaldehyde for 10 min. Using a Raman microscope, multiple cells were scanned to obtain SERS images and spectral data, and the average value was taken to represent the SERS results. The excitation wavelength was 633 nm, the power was 2.5 mW, the laser spot diameter was 1 μm, the exposure time was 1 s, and the SERS imaging and spectral results were as follows: Figure 14-15 As shown. Fig.14 It can be seen that with the increase of intracellular ROS concentration, at 1650cm -1 The SERS signal at the Fig.15 The spectral results are consistent with those of .

[0079] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A dual-mode nanoprobe, characterized in that: The dual-mode nanoprobe includes gold core-silver shell nanoparticles, and a protective agent and a polypeptide chain respectively modified on the surface of the gold core-silver shell nanoparticles; the polypeptide chain includes a mitochondrial targeting peptide, and a thiol group and a fluorescent molecule respectively modified on both ends of the mitochondrial targeting peptide; the gold core-silver shell nanoparticles and the polypeptide chain are connected by a silver-sulfur covalent bond; the sequence of the mitochondrial targeting peptide is MLALLGWWWFFSRKKC.

2. The dual-mode nanoprobe according to claim 1, characterized in that: The particle size of the gold core-silver shell nanoparticles is 18nm-24nm; the thickness of the silver layer is 4nm-5nm.

3. The dual-mode nanoprobe according to claim 1, characterized in that: The protective agent is selected from methoxy polyethylene glycol thiol and / or Tween-20.

4. The dual-mode nanoprobe according to claim 1, characterized in that: The molar concentration ratio of the gold core-silver shell nanoparticles, the protective agent and the polypeptide chain is 1:(2500-5000):(1200-1600).

5. The dual-mode nanoprobe according to claim 1, characterized in that: The fluorescent molecule is selected from one or more of rhodamine B, fluorescein isothiocyanate, carboxyfluorescein and anthocyanin fluorescent dyes.

6. The method for preparing a dual-mode nanoprobe according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: adding a protective agent solution and a polypeptide chain solution to a gold core-silver shell nanoparticle solution in sequence for incubation to obtain the dual-mode nanoprobe.

7. The method for preparing the dual-mode nanoprobe according to claim 6, characterized in that: The concentration of the gold core-silver shell nanoparticle solution is 2 nM; the concentration of the protective agent solution is 0.1 mM-0.2 mM; and the concentration of the polypeptide chain solution is 240 μM-320 μM.

8. The method for preparing a dual-mode nanoprobe according to claim 6, characterized in that: The volume ratio of the gold core-silver shell nanoparticle solution, the protective agent solution, and the polypeptide chain solution is 1000:(50-100):(10-20).

9. Use of the dual-mode nanoprobe according to any one of claims 1 to 5 in ROS detection.

10. The use according to claim 9, characterized in that: The concentration of the dual-mode nanoprobe in the application is 0.25 nM-1 nM.

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