Oxygen-sensitive fluorescent nanoprobe as well as preparation method and application thereof

Abstract

The invention relates to an oxygen-sensitive fluorescent nanoprobe as well as a preparation method and application thereof. The fluorescent nanoprobe has a unique core / shell structure, and a hydrophobic oxygen-sensitive dye with unmatched chemical properties and a hydrophilic silicon dioxide material are fused to prepare the fluorescent nanoprobe which is high in brightness, good in light stability, high in sensitivity, good in reversibility, good in biocompatibility and easy in surface modification. The real-time and dynamic analysis of the oxygen consumption rate, the cell energy metabolism and the mitochondrial function in a single living cell is realized. By designing and synthesizing the high-performance fluorescent nanoprobe, the problem that energy metabolism analysis of a single living cell cannot be realized at a single cell level is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomaterials, and in particular relates to an oxygen-sensitive fluorescent nano-probe and a preparation method and application thereof. Background Art

[0002] Energy metabolism is the key to the survival of any living organism. Dysfunction of energy metabolism has been repeatedly confirmed to be closely related to major diseases such as cardiovascular and cerebrovascular diseases, cancer, and Alzheimer's disease in humans. Due to the existence of cellular heterogeneity, studying the energy metabolism of individual living cells at the single-cell level will provide a new perspective for a deeper understanding of basic biological events such as why stem cells can differentiate into organs with completely different energy consumption, how normal cells transform into tumor cells, the origin of drug resistance, and aging. In addition, measuring the energy metabolism of living cells at the single-cell level will also provide a powerful tool for understanding disease progression, drug development, patient treatment, and biomedical research.

[0003] However, measuring the energy metabolism of a single living cell faces huge challenges. First, the research object is small in size, the amount of sample that can be obtained is very small (picolitre level), the composition is complex and variable, and any disturbance will affect the metabolism of the research object, which may affect the quality of the measurement data or even cause cell apoptosis. Therefore, it is necessary to develop probes of small enough size to obtain energy metabolism data of living cells in normal activity without affecting the normal metabolism of cells. This requires the development of detection materials and technologies with high sensitivity, high selectivity, high stability and good biocompatibility, which undoubtedly puts extremely high demands on the development of new materials and measurement methods.

[0004] Before 1953, because the measurement technology was far less developed than it is now, researchers could not measure the energy metabolism of cells in a single living cell. In order to measure the energy metabolism of cells, people had to culture a batch of cells, and after the number of cells accumulated enough, they separated and purified the mitochondria in the cells, and then tested the function of the purified mitochondria. The experiment included using the Winkler titration method to measure the oxygen consumption rate in the solution, and using the enzyme reaction method to quantify the ATP production rate, so as to evaluate the energy metabolism state of the mitochondria. Obviously, the mitochondria separated in vitro cannot represent their activity level in living cells. At the same time, because the number and function of the separated mitochondria are very different from those in living cells, it is difficult to truly reflect the energy metabolism state of living cells. After the 1950s, with the invention of the oxygen electrode by Clark and the popularization of the pH electrode, people finally had the opportunity to measure the oxygen consumption rate OCR of the cell population and the acidification rate ECAR of the cell culture medium without destroying the cells, thereby characterizing the energy metabolism state of the living cell population. The development of fluorescence sensing technology in the early 21st century has further promoted the rapid development of this field. Scientists have used the characteristics of fluorescence sensing technology that can be easily miniaturized to create equipment that can measure the energy metabolism of living cell groups in multi-well plates. Representative instruments and equipment include Oroboros' O2K Cell Energy Metabolism Analysis System and Agilent's Seahorse Cell Energy Metabolism Analyzer. The invention of these instruments has greatly promoted the development of the field of living cell energy metabolism measurement. However, due to the limitations of the measurement method, these instruments need to measure the comprehensive energy metabolism results of a group composed of tens of thousands of cells, which obviously ignores the heterogeneity of cells and cannot measure the energy metabolism state of living cells at the single cell level.

[0005] The rapid development of fluorescence imaging technology in recent years naturally has the ability to distinguish single cells. Since the 1990s, fluorescent dyes have been shining in the field of living cell labeling and qualitative analysis. However, since most fluorescent dyes will experience severe photobleaching under strong light from a microscope, they will completely lose fluorescence within a few seconds, making it difficult for fluorescent dyes to achieve quantitative analysis in living cells. In addition, fluorescent dyes usually have a conjugated water-transporting luminescent core, and their hydrophobicity is not compatible with the hydrophilic environment in the cell. It is easy for fluorescent dyes to aggregate, causing their luminescent properties to change, making it impossible to achieve in vitro calibration of the measured results. Wang et al. reviewed oxygen-sensitive probes for oxygen measurement in 2014 (Chem. Soc. Rev. 2014, 43, 3666-3761.). Among the many oxygen-sensitive probes reported, metal-organic complexes have the advantages of large Stokes shift, high sensitivity, and long fluorescence lifetime. The fluorescence signal is easily separated from the excitation light signal, and is an ideal fluorescent oxygen-sensitive probe. However, most metal-organic complexes have strong hydrophobicity and are difficult to be directly used in the study of living cells. In most cases, these oxygen-sensitive dyes are either encapsulated in a polymer matrix to form a polymer nanoprobe for oxygen sensing; or by chemical synthesis, hydrophilic Dendrimer polymers are used to modify their hydrophobic luminescent cores (ACS Applied Materials & Interfaces 2009, 1, 1292-1304.). The polymers used to encapsulate oxygen-sensitive probes are usually chemically inert and difficult to further functionalize, and their distribution in cells cannot be effectively controlled. A large number of research results show that fluorescent nanoprobes based on silica usually have the characteristics of good optical properties, high brightness, good stability, etc., and have good biocompatibility. Its excellent chemically controllable modification ability makes silica materials have broad biological application prospects, and silica nanoparticles doped with fluorescent dyes have even been used in human trials (Science Translational Medicine 2014, 6, 260ra149.). Wang et al.'s recent study found that silica nanoparticles have good gas permeability and can be used to construct oxygen-sensitive nanoprobes (Analytical Chemistry 2019, 91, 15625-15633.); however, metal-organic complex oxygen-sensitive probes with good photostability are difficult to construct oxygen-sensitive nanoprobes by simple physical embedding. Congreve et al. tried to use oleic acid to encapsulate hydrophobic metal-organic complex oxygen-sensitive dyes inside silica nanoparticles, but because the double bonds of unsaturated fatty acids are reducing, the nanomaterials prepared by this method have no oxygen response (Nature, 2022, 604, 474-478.).Silica nanoparticles are regarded as potential carriers due to their good optical properties, stability, biocompatibility and chemically controllable modification capabilities, but how to effectively encapsulate metal-organic complex oxygen-sensitive probes with good photostability into silica nanoparticles remains a difficult problem. Summary of the invention

[0006] The main purpose of the present invention is to provide an oxygen-sensitive fluorescent nanoprobe and its preparation method and application, which has the characteristics of high luminescence brightness, good photostability, easy surface modification, good biocompatibility, high sensitivity, good reversibility, etc., and can meet the needs of energy metabolism analysis of single living cells. Through a specific preparation process, the present invention solves the limitations of existing oxygen-sensitive probes in living cell applications, such as photobleaching, hydrophobicity, and uncontrollable in vivo distribution.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing an oxygen-sensitive fluorescent nanoprobe. The method cleverly dissolves a hydrophobic oxygen-sensitive dye in a saturated fatty acid, which serves as the hydrophobic core of the nanoprobe, providing a good hydrophobic environment for the dye, and then constructs a fluorescent nanoprobe with a unique core-shell structure through a series of delicate chemical steps.

[0009] The method for preparing the oxygen-sensitive fluorescent nanoprobe comprises the following steps:

[0010] 1) dissolving a hydrophobic oxygen-sensitive dye in a saturated fatty acid to form a mixture A;

[0011] 2) Mixing mixture A and ultrapure water, and stirring vigorously until a uniform emulsion B is formed;

[0012] 3) mixing the positively charged siloxane reagent and the emulsion B to form a transparent microemulsion C under stirring;

[0013] 4) adding a protective colloid to the microemulsion C to obtain a stabilized mixed solution D; wherein the protective colloid has the function of preventing collision and agglomeration between nanoparticles and promoting uniform dispersion of the nanoparticles;

[0014] 5) mixing the negatively charged siloxane reagent, the orthosilicate and the mixed solution D to obtain a mixed solution E;

[0015] 6) The mixed solution E is continuously stirred and reacted at 20-100° C. to form a silica shell layer. After the reaction is completed, the product is centrifuged and washed.

[0016] If the saturated fatty acid is solid, it can be made liquid by heating, so that the hydrophobic oxygen-sensitive dye can be dissolved therein to form a mixture A.

[0017] Specifically, the present invention forms a grease-like mixture A by mixing a hydrophobic oxygen-sensitive dye with a saturated fatty acid, and then disperses the mixture A in water to form a microemulsion B, and then stabilizes the microemulsion B with a positively charged siloxane reagent to form a transparent water-in-oil microemulsion C. After adding a protective colloid to further stabilize the microemulsion C to form a mixed solution D, a negatively charged siloxane reagent and an orthosilicate are added to obtain a mixed solution E, and the orthosilicate is hydrolyzed and condensed in the mixed solution E to form a silica shell to fix the droplets to form an oxygen-sensitive fluorescent nanoprobe. When adding the orthosilicate, the negatively charged siloxane reagent is added simultaneously, which can effectively increase the surface charge between the nanoprobes, increase the electrostatic repulsion between the nanoparticles, and is conducive to the formation of a monodispersed nanoprobe. The method described in the present invention can successfully prepare a high-performance oxygen-sensitive fluorescent nanoprobe with high brightness, good photostability, fast response speed, and high sensitivity to oxygen.

[0018] As an example, in step 1), the mass ratio of the hydrophobic oxygen-sensitive dye to the saturated fatty acid is (0.1-3):100.

[0019] As an example, in step 2), the volume ratio of the mixture A to ultrapure water is 1:(50-200).

[0020] As an example, in step 3), the volume ratio of the positively charged siloxane reagent to the emulsion B is 1:(150-350).

[0021] As an example, in step 4), the mass ratio of the protective colloid to the microemulsion C is 1:(10-100).

[0022] As an example, in step 5), the volume ratio of the negatively charged siloxane reagent, the orthosilicate, and the mixed solution D is 1:(5-20):(30-100).

[0023] The hydrophobic oxygen-sensitive dye includes, but is not limited to, a porphyrin or porphine complex oxygen-sensitive probe of a hydrophobic metal platinum, palladium, ruthenium, or iridium. As an example, the hydrophobic metal organic complex is selected from one of platinum (II) meso-tetrakis (pentafluorobenzene) porphine, platinum octaethyl porphyrin, palladium octaethyl porphyrin, tetraphenyl-tetrabenzoporphyrin platinum, and tetraphenyl-tetrabenzoporphyrin palladium complexes.

[0024] Preferably, the number of carbon atoms in the carbon chain of the saturated fatty acid is 6 to 20. As an example, the saturated fatty acid is selected from one of stearic acid, lauric acid and nonanoic acid.

[0025] The positively charged silane reagent includes but is not limited to 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminobutyltriethoxysilane and the like.

[0026] According to a specific embodiment of the present invention, the protective colloid is polyvinyl pyrrolidone. The protective colloid does not exclude other materials, as long as it has the function of avoiding collision and agglomeration between nanoparticles and promoting uniform dispersion of nanoparticles, thereby helping to control the particle size and dispersibility of nanoparticles. Preferably, the viscosity K value of polyvinyl pyrrolidone in water is between 12 and 120 to further optimize the particle size dispersibility and particle size of the nanoprobe.

[0027] The negatively charged siloxane reagent includes, but is not limited to, 3-(trihydroxysilyl)propylmethylphosphonic acid sodium salt, 3-(trihydroxysilyl)-propanesulfonic acid, triethoxysilylpropylmaleic acid, and the like.

[0028] As an example, the orthosilicate is one of tetramethoxysilane, tetraethyl orthosilicate, propyl orthosilicate, and butyl orthosilicate.

[0029] Optionally, the method for preparing the nanoprobe further comprises step 7): modifying the surface of the nanoparticles obtained after washing in step 6) with other fluorescent probes or organelle or sub-organelle targeting groups. By modifying the surface of the nanoprobe with cell or sub-organelle targeting groups, the nanoprobe can be accurately positioned on the cell or sub-organelle to achieve real-time, quantitative measurement of oxygen concentration in living cells. By using inhibitors to regulate mitochondrial function, the oxygen-sensitive probe described in the present invention was successfully used to measure the oxygen consumption rate OCR in a single living cell and to draw a cellular energy metabolism map.

[0030] The present invention also provides an oxygen-sensitive fluorescent nanoprobe, which has obvious core-shell structural characteristics, with a saturated fatty acid hydrophobic core inside, embedded with a hydrophobic oxygen-sensitive dye; the outer layer is a hydrophilic silica shell. Silica material has good gas permeability and excellent biocompatibility. By embedding the oxygen-sensitive probe inside the nanoparticles, the interference of the external environment on the fluorescence quantitative analysis can be effectively reduced, and the accuracy of oxygen detection can be improved. The prepared fluorescent nanoprobe has excellent photostability, good surface modifiability and biocompatibility; combined with fluorescence imaging technology, the use of this nanoprobe can realize the energy metabolism analysis of single living cells.

[0031] Optionally, the hydrophilic silica shell layer is modified with other fluorescent probes, cell targeting groups or subcellular organelle targeting groups on its surface by covalent bonding.

[0032] The present invention also provides the application of the oxygen-sensitive fluorescent nanoprobe described above in the detection of oxygen concentration in living cells and the analysis of energy metabolism in living cells.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. The oxygen-sensitive fluorescent nanoprobe prepared by the present invention integrates the hydrophobic oxygen-sensitive dye and the hydrophilic silica material with mismatched chemical properties into the same nanoparticle; the nanoprobe has a unique core-shell structure, excellent photostability, good oxygen response sensitivity and reversibility, good biocompatibility and surface modifiability. Combined with fluorescence imaging technology, the nanoprobe can realize real-time measurement of intracellular oxygen concentration of single living cells and be used to analyze the energy metabolism state of cells.

[0035] 2. The hydrophobic oxygen-sensitive dye dissolved in saturated fatty acids is located in the core of the nanoprobe, which prevents the dye from being disturbed by the external environment. It has extremely high photostability and improves the accuracy of detection. At the same time, the design of the core / shell structure also avoids aggregation, quenching and leakage of the oxygen-sensitive dye.

[0036] 3. A layer of silica shell grows on the outside of the fatty acid, which not only strengthens the structure of the entire material, but also gives the nanoprobe good biocompatibility. Other fluorescent probes or organelle targeting groups can be modified on its surface, which is conducive to the further functionalization of the nanoprobe.

[0037] 4. When the oxygen-sensitive fluorescent nanoprobe is exposed to different oxygen concentration atmospheres and irradiated with appropriate excitation light, the fluorescence intensity and fluorescence lifetime of the nanoprobe will change significantly. It can be used for long-term continuous and accurate monitoring of oxygen concentration in living cells. Combined with fluorescence imaging technology, it can realize real-time measurement of the energy metabolism state of a single living cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . Transmission electron microscopy image of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1.

[0039] Figure 2 .(a) Changes in fluorescence intensity of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1 under different oxygen concentration gas atmospheres; (b) Stern-Volmer curve of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1.

[0040] Figure 3 .Photostability test curve of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1.

[0041] Figure 4 .The curve of the fluorescence intensity of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1 changing over time under the cyclic switching of the anaerobic-air atmosphere.

[0042] Figure 5.The oxygen consumption rate diagram of a single PC12 cell treated with a mitochondrial inhibitor measured by the oxygen-sensitive fluorescent nanoprobe prepared in Example 1 (blue bar) and the oxygen consumption rate diagram of PC12 cells (cell number: 20,000) treated with a mitochondrial inhibitor measured by the Agilent Seahorse XFe96 cell energy analyzer (red bar). DETAILED DESCRIPTION

[0043] The present invention will be further described below by way of examples.

[0044] Example 1

[0045] 1) Weigh 0.84 mg of platinum (II) meso-tetrakis(pentafluorophenyl)porphine and dissolve it in 1.0 mL of liquid stearic acid, and stir at 70°C to form a red transparent mixture A; the mass ratio of the dye to stearic acid is 0.1:100;

[0046] 2) Then, 100 μL of mixture A was added to 20.0 mL of ultrapure water and stirred vigorously at 70° C. until a uniform emulsion B was formed; the volume ratio of mixture A to ultrapure water was 1:200;

[0047] 3) Add 134 μL of 3-aminopropyltrimethoxysilane to emulsion B and form a transparent

[0048] The volume ratio of 3-aminopropyltrimethoxysilane to emulsion B is 1:150;

[0049] 4) adding 200.0 mg of polyvinyl pyrrolidone K-90 to the microemulsion C to stabilize the microemulsion C, thereby obtaining a mixed solution D; the mass ratio of polyvinyl pyrrolidone K-90 to the microemulsion C is 1:100;

[0050] 5) Add 200 μL of 3-(trihydroxysilyl)propylmethylphosphonic acid sodium salt and 1.0 mL of

[0051] Tetraethyl orthosilicate to obtain mixed solution E; 3-(trihydroxysilyl)propylmethylphosphonic acid sodium salt, tetraethyl orthosilicate, and mixed solution D in a volume ratio of 1:5:100;

[0052] 6) Stirring the mixed solution E at 70° C. for 48 hours;

[0053] 7) The formed nanoparticle suspension was cooled to room temperature and washed by centrifugation with ultrapure water at a speed of 9000g.

[0054] Oxygen-sensitive fluorescent nanoprobes were obtained 3 times.

[0055] Figure 1This is a transmission electron microscope image of the oxygen-sensitive fluorescent nanoprobe prepared as above. The prepared fluorescent nanoprobe has a small nanoparticle size, with an average nanoparticle size of 174 nm.

[0056] Example 2

[0057] 1) Weigh 26.49 mg of tetraphenyl-tetrabenzoporphyrin palladium complex and dissolve it in 1.0 mL of liquid lauric acid, and stir at 45° C. to form a green transparent mixture A; the mass ratio of the dye to the lauric acid is 3.0:100;

[0058] 2) Then, 400 μL of mixture A was added to 20.0 mL of ultrapure water and stirred vigorously at 50° C. until a uniform emulsion B was formed; the volume ratio of mixture A to ultrapure water was 1:50;

[0059] 3) Add 100.5 μL of 3-aminopropyltriethoxysilane to emulsion B, and form a transparent

[0060] The volume ratio of 3-aminopropyltriethoxysilane to emulsion B is 1:200;

[0061] 4) adding 800.0 mg of polyvinyl pyrrolidone K-60 to the microemulsion C to stabilize the microemulsion C, thereby obtaining a mixed solution D; the mass ratio of polyvinyl pyrrolidone K-60 to microemulsion C is 1:25;

[0062] 5) adding 400 μL of 3-(trihydroxysilyl)-propanesulfonic acid and 2.0 mL of tetramethoxysilane to the mixed solution D to obtain a mixed solution E; the volume ratio of 3-(trihydroxysilyl)-propanesulfonic acid, tetramethoxysilane, and the mixed solution D is 1:5:50;

[0063] 6) Stirring the mixed solution E at 60° C. for 36 hours;

[0064] 7) The formed nanoparticle suspension was cooled to room temperature, and washed three times by centrifugation with ultrapure water at 9000 g to obtain an oxygen-sensitive fluorescent nanoprobe.

[0065] Example 3

[0066] 1) Weigh 0.906 mg of platinum octaethylporphyrin and dissolve it in 1.0 mL of nonanoic acid, and stir at room temperature to form a red transparent mixture A; the mass ratio of the dye to nonanoic acid is 1.0:100;

[0067] 2) Then, 200 μL of mixture A was added to 20.0 mL of ultrapure water and stirred vigorously at room temperature until a uniform emulsion B was formed; the volume ratio of mixture A to ultrapure water was 1:100;

[0068] 3) Add 202.0 μL of 3-aminobutyltriethoxysilane to emulsion B, and a transparent

[0069] Bright pink microemulsion C; the volume ratio of 3-aminobutyltriethoxysilane to emulsion B is 1:100;

[0070] 4) adding 2000.0 mg of polyvinyl pyrrolidone K-30 to the microemulsion C to stabilize the microemulsion C, thereby obtaining a mixed solution D; the mass ratio of polyvinyl pyrrolidone K-30 to microemulsion C is 1:10;

[0071] 5) Add 666.6 μL of triethoxysilylpropyl maleic acid and 3.3 mL of tetraethyl orthosilicate to the mixed solution D to obtain a mixed solution E; the volume ratio of triethoxysilylpropyl maleic acid, tetraethyl orthosilicate, and the mixed solution D is 1:5:30;

[0072] 6) Stirring the mixed solution E at 45°C for 24 hours;

[0073] 7) The formed nanoparticle suspension was cooled to room temperature, and washed three times with ultrapure water by centrifugation at 9000 g to obtain an oxygen-sensitive fluorescent nanoprobe.

[0074] Experiment 1: Analysis of fluorescence intensity responsiveness of nanoprobes in atmospheres with different oxygen concentrations

[0075] 1) Prepare nitrogen and oxygen supply sources;

[0076] 2) Adjust the gas flow meter to keep the total flow rate of nitrogen and oxygen constant, and prepare gas atmospheres with different oxygen concentrations by adjusting the flow rate ratio of the two;

[0077] 3) Pass the prepared gas into the cuvette containing the oxygen-sensitive fluorescent nanoprobe, and start measuring the fluorescence intensity after stabilizing for a period of time;

[0078] 4) Record the fluorescence intensity values ​​under different oxygen concentrations;

[0079] 5) According to the Stern-Volmer equation, the I / I value at each oxygen concentration is calculated, where I represents the fluorescence intensity under a nitrogen-saturated gas atmosphere and I represents the fluorescence intensity at the current oxygen concentration. The Stern-Volmer curve is drawn with I / I as the ordinate and the volume percentage of oxygen content as the abscissa.

[0080] Figure 2 (a) is the fluorescence intensity change of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1 in gas atmospheres with different oxygen concentrations, Figure 2 (b) is the Stern-Volmer curve of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1. Figure 2 (a) and Figure 2 (b) It can be seen that the fluorescence intensity of the nanoprobe decreases in an oxygen concentration-dependent manner.

[0081] Experiment 2 Evaluation of the photostability of nanoprobes

[0082] 1) Weigh 10.0 mg of oxygen-sensitive fluorescent nanoprobe, add it to 1.0 mL of ultrapure water, and disperse it evenly by ultrasonication;

[0083] 2) Take 200 μL of the mixed solution and drop it on the surface of the PET plastic film;

[0084] 3) Place the film in a 35°C oven to dry the water naturally;

[0085] 4) The dried film was placed in a cuvette, and the photostability of the oxygen-sensitive fluorescent nanoprobe was tested in air using a fluorescence spectrometer, with an excitation wavelength of 390 nm, an emission wavelength of 650 nm, an excitation slit width of 20 nm, and an emission slit width of 5 nm.

[0086] Figure 3 This is a photostability test curve of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1. After continuous irradiation in air for 2 hours, its fluorescence intensity only decreased by 4.71%.

[0087] Experiment 3 Verification of the reversibility of the nanoprobe oxygen response

[0088] 1) Weigh 10.0 mg of oxygen-sensitive fluorescent nanoprobe, add it to 1.0 mL of ultrapure water, and disperse it evenly by ultrasonication;

[0089] 2) Take 200 μL of the mixed solution and drop it on the surface of the PET plastic film;

[0090] 3) Place the film in a 35°C oven to dry the water naturally;

[0091] 4) The dried film was placed in a cuvette, and the reversibility of oxygen response of the oxygen-sensitive fluorescent nanoprobe was tested by switching between an oxygen-free and air atmosphere using a fluorescence spectrometer, with an excitation wavelength of 390 nm and an emission wavelength of 650 nm.

[0092] Figure 4 The graph shows the change of fluorescence intensity over time of the oxygen-sensitive fluorescent nanoprobe prepared according to Example 1 under cyclic switching between an oxygen-free and air atmosphere.

[0093] Depend on Figure 4 The results show that the fluorescence intensity of the nanoprobe shows good oxygen response sensitivity and reversibility as it switches back and forth between oxygen-free atmosphere and air atmosphere.

[0094] Experiment 4: Using nanoprobes to detect oxygen concentration in living cells and analyze energy metabolism status

[0095] 1) PC12 cells (2×10 5 cells / well) were seeded into 24-well plates and maintained in DMEM medium.

[0096] Cultivate for 24 hours;

[0097] 2) After incubating the oxygen-sensitive fluorescent nanoprobe (200 μg / mL) with PC12 cells for 12 hours, the oxygen-sensitive fluorescent nanoprobe that was not absorbed by the cells was washed away with Hank's buffer;

[0098] 3) Then add 200 μL of fresh DMEM medium to each well;

[0099] 4) Carefully add 500 μL of mineral oil to each well to form a thin gas barrier layer;

[0100] The above steps are for the control group. When using inhibitors, the operation steps are as follows:

[0101] 1) PC12 cells (2×10 5 cells / well) were seeded into 24-well plates and cultured in DMEM medium for 24 h;

[0102] 2) After incubating the oxygen-sensitive fluorescent nanoprobe (200 μg / mL) with PC12 cells for 12 hours, the oxygen-sensitive fluorescent nanoprobe that was not absorbed by the cells was washed away with Hank's buffer;

[0103] 3) dissolving the inhibitor in DMSO to form an inhibitor-containing DMSO solution with an inhibitor concentration of 1.0 mM;

[0104] 4) Take 50.0 μL of the DMSO solution containing the inhibitor and mix it with 9.95 mL of DMEM medium to form a DMEM medium containing 5.0 μM inhibitor;

[0105] 5) Add 200 μL of DMEM medium containing 5.0 μM inhibitor to the 24-well plate, and add 500 μL of mineral oil on top of the medium to form a gas blocking layer;

[0106] 6) Use fluorescence lifetime imaging microscopy to record the luminescence lifetime of oxygen-sensitive fluorescent nanoprobes for 180

[0107] The measurement interval is 10 minutes.

[0108] The changing trend of oxygen concentration in PC12 cells was calculated based on the standard working curve of oxygen-sensitive fluorescent nanoprobe (control group) and the change of its luminescence lifetime (when using inhibitor), and then the amount of oxygen consumed in the first minute was calculated;

[0109] The cell energy metabolism analysis experiment was carried out according to the instructions of the Seahorse XFe96 cell energy metabolism analysis instrument, and the data thereof were compared with the data of the present invention.

[0110] The above inhibitor is a mitochondrial inhibitor, specifically rotenone in this experiment.

[0111] Figure 5 The oxygen consumption rate diagram of a single PC12 cell treated with a mitochondrial inhibitor measured by the oxygen-sensitive fluorescent nanoprobe (blue bar) prepared according to Example 1 and the oxygen consumption rate diagram of PC12 cells (cell number: 20,000) treated with a mitochondrial inhibitor measured by the Agilent Seahorse XFe96 Cell Energy Analyzer (red bar). By using inhibitors to regulate mitochondrial function, the oxygen-sensitive probe described in the present invention was successfully used to measure the oxygen consumption rate OCR in a single living cell.

Claims

1. An oxygen-sensitive fluorescent nanoprobe, characterized in that: The oxygen-sensitive fluorescent nanoprobe has obvious core-shell structural characteristics. Its interior is a saturated fatty acid hydrophobic core, which is embedded with a hydrophobic oxygen-sensitive dye; its outer layer is a hydrophilic silica shell.

2. The nanoprobe according to claim 1, characterized in that The hydrophilic silica shell layer can be modified with other fluorescent probes, cell targeting groups or subcellular organelle targeting groups on its surface by covalent bonding.

3. A method for preparing an oxygen-sensitive fluorescent nanoprobe, characterized in that: The following steps are involved: 1) dissolving a hydrophobic oxygen-sensitive dye in a saturated fatty acid to form a mixture A; 2) Mixing mixture A and ultrapure water, and stirring vigorously until a uniform emulsion B is formed; 3) mixing the positively charged siloxane reagent and the emulsion B to form a transparent microemulsion C under stirring; 4) adding a protective colloid to the microemulsion C to obtain a stabilized mixed solution D; wherein the protective colloid has the function of preventing collision and agglomeration between nanoparticles and promoting uniform dispersion of the nanoparticles; 5) mixing the negatively charged siloxane reagent, the orthosilicate and the mixed solution D to obtain a mixed solution E; 6) The mixed solution E is continuously stirred and reacted at 20-100° C. to form a silica shell layer. After the reaction is completed, the product is centrifuged and washed.

4. The preparation method according to claim 3, characterized in that: The carbon chain of the saturated fatty acid has 6 to 20 carbon atoms.

5. The preparation method according to claim 3, characterized in that: In step 1), the mass ratio of the hydrophobic oxygen-sensitive dye to the saturated fatty acid is (0.1-3):100; in step 2), the volume ratio of the mixture A to ultrapure water is 1:(50-200); in step 3), the volume ratio of the positively charged siloxane reagent to emulsion B is 1:(150-350); in step 4), the mass ratio of the protective colloid to the microemulsion C is 1:(10-100); in step 5), the volume ratio of the negatively charged siloxane reagent, orthosilicate, and mixed solution D is 1:(5-20):(30-100).

6. The preparation method according to claim 3, characterized in that: The protective colloid is polyvinyl pyrrolidone.

7. The preparation method according to claim 3, characterized in that: The hydrophobic oxygen-sensitive dye is selected from one of the porphyrin or porphine complex oxygen-sensitive probes of metal platinum, palladium, ruthenium, and iridium.

8. The preparation method according to claim 3, characterized in that: The positively charged silane reagent is selected from one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane and 3-aminobutyltriethoxysilane.

9. The preparation method according to claim 3, characterized in that: The negatively charged siloxane reagent is selected from one of 3-(trihydroxysilyl)propylmethylphosphonic acid sodium salt, 3-(trihydroxysilyl)-propanesulfonic acid, and triethoxysilylpropylmaleic acid.

10. Use of the oxygen-sensitive fluorescent nanoprobe according to claim 1 or 2 in the detection of oxygen concentration in living cells and the analysis of energy metabolism in living cells.

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