An oxygen-sensitive fluorescent nanoprobe, a preparation method and application thereof

Abstract

The present application relates to a kind of oxygen-sensitive fluorescent nanoprobes and its preparation method and application.The fluorescent nanoprobes have unique core / shell structure, and the hydrophobic oxygen-sensitive dye with incompatible chemical properties is fused with hydrophilic silica material, to prepare a kind of fluorescent nanoprobes with high brightness, good light stability, high sensitivity, good reversibility, good biocompatibility and easy surface modification, to realize the real-time, dynamic analysis of oxygen consumption rate, cell energy metabolism and mitochondrial function in single living cell.The present application solves the problem that single living cell energy metabolism analysis cannot be realized at single cell level by designing and synthesizing high-performance fluorescent nanoprobes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiomaterials, and particularly relates to an oxygen-sensitive fluorescent nanoprobe, a preparation method and application thereof. BACKGROUND

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

[0003] However, there are great challenges in measuring the energy metabolism of a single living cell. First, the research object is small in size, and the amount of sample that can be obtained is extremely small (picoliter level), the composition is complex and variable, and any disturbance will affect the metabolism of the research object. Lightly, it will affect the quality of the measurement data, and heavily, it will lead to cell apoptosis. Therefore, it is necessary to develop a probe with a small enough size to obtain the energy metabolism data of living cells under normal activity without affecting the normal metabolism of the cells, which requires the development of detection materials and technologies with high sensitivity, high selectivity, high stability and good biocompatibility. This undoubtedly puts forward very high requirements for the development of new materials and measurement methods.

[0004] Before 1953, due to the measurement technology far less developed than 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, wait until the number of cells accumulated enough, then separate and purify the mitochondria in the cells, and then test the function of the purified mitochondria, including measuring the oxygen consumption rate in solution using Winkler titration method, using enzyme reaction method to quantify the rate of ATP production, so as to evaluate the energy metabolism state of mitochondria. Obviously, the isolated mitochondria ex vivo cannot represent the activity level in living cells, and at the same time, due to the difference in the number and function of isolated mitochondria from living cells, it is difficult to truly reflect the energy metabolism state of living cells. After 1950s, with the invention of oxygen electrode by Clark and the popularity of pH electrode, people finally had the opportunity to measure the oxygen consumption rate OCR and the acidification rate ECAR of cell culture solution without destroying the cells, and then characterize the energy metabolism state of living cell population. The development of fluorescence sensing technology in the early 21st century further promoted the rapid development of this field. Scientists took advantage of the easy miniaturization of fluorescence sensing technology to manufacture equipment that can measure the energy metabolism of living cell population in a multi-well plate. Representative instruments include O2K cell energy metabolism analysis system of Oroboros company and Seahorse cell energy metabolism analyzer of Agilent company. The invention of these instruments has greatly promoted the development of living cell energy metabolism measurement field. However, due to the limitations of measurement methods, these instruments need to measure the comprehensive energy metabolism results of thousands of cells, obviously ignoring the heterogeneity of cells, and cannot realize the measurement of energy metabolism state of living cells at single cell level.

[0005] The rapid development of fluorescence imaging technology in recent years has the ability to distinguish single cells naturally. Fluorescent dyes have been widely used in live cell labeling and qualitative analysis since the 1990s. However, most fluorescent dyes will undergo severe photobleaching under strong light, losing fluorescence completely within a few seconds, making it difficult to perform quantitative analysis in live cells. In addition, fluorescent dyes usually have a hydrophobic light-emitting core, which cannot be compatible with the hydrophilic environment in cells. The aggregation of fluorescent dyes will change their light-emitting properties, making it impossible to calibrate the results in vitro. 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, making it easy to separate the fluorescence signal from the excitation light signal, making it an ideal fluorescent oxygen-sensitive probe. However, most metal-organic complexes have strong hydrophobicity and cannot be directly used for live cell research. In most cases, these oxygen-sensitive dyes are either encapsulated in a polymer matrix to form a polymer nanoprobe for oxygen sensing or modified with hydrophilic Dendrimer polymers to modify their hydrophobic light-emitting core through chemical synthesis (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. Numerous studies have shown that silica-based fluorescent nanoprobes usually have good optical properties, high brightness, and good stability, and have good biocompatibility. The excellent chemical controllable modification ability of silica materials makes them 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. recently 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 light stability are difficult to construct oxygen-sensitive nanoprobes through simple physical embedding. Congreve et al. tried to use oleic acid to encapsulate hydrophobic metal-organic complex oxygen-sensitive dyes inside silica nanoparticles, but the double bonds of unsaturated fatty acids have reducing properties, and the nanomaterials prepared by this method have no oxygen response (Nature, 2022, 604, 474-478.).Silica nanoparticles are considered as potential carriers due to their good optical properties, stability, biocompatibility and chemically controllable modification ability, but it is still a difficult problem how to effectively encapsulate the metal-organic complex oxygen-sensitive probe with good light stability in silica nanoparticles. SUMMARY

[0006] The main purpose of the present application is to provide an oxygen-sensitive fluorescent nanoprobe, a preparation method and application thereof, which has the characteristics of high luminous brightness, good light stability, easy surface modification, good biocompatibility, high sensitivity and good reversibility, and can meet the needs of single living cell energy metabolism analysis. Through a specific preparation process, the present application 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 purpose, the technical scheme of the present application is as follows:

[0008] The present application provides a preparation method of an oxygen-sensitive fluorescent nanoprobe. The method ingeniously dissolves a hydrophobic oxygen-sensitive dye in saturated fatty acid to form a mixture A, which provides a good hydrophobic environment for the dye, and then a series of fine chemical steps are used to construct a fluorescent nanoprobe with a unique core-shell structure.

[0009] The preparation method of the oxygen-sensitive fluorescent nanoprobe comprises the following steps:

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

[0011] 2) Mix the mixture A and ultrapure water, and stir vigorously to form a uniform emulsion B;

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

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

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

[0015] 6) Stir the mixed solution E continuously at 20-100 DEG C to form a silica shell layer, and then centrifuge and wash the product after the reaction is completed.

[0016] If the saturated fatty acid is solid, it can be heated to make it liquid, so that the hydrophobic oxygen-sensitive dye is dissolved in it to form the mixture A.

[0017] Specifically, the present application forms an oil-like mixture A by mixing a hydrophobic oxygen-sensitive dye with a saturated fatty acid, then disperses the mixture A in water to form a microemulsion B, and then forms a transparent oil-in-water microemulsion C by stabilizing the microemulsion B with a positively charged siloxane reagent. After adding a protective colloid to further stabilize the microemulsion C to form a mixture D, a negatively charged siloxane reagent and a positive silicate are added to form a mixture E. The liquid droplets are fixed by using the positive silicate to hydrolyze and condense in the mixture E to form a silica shell layer, thereby forming an oxygen-sensitive fluorescent nanoprobes. The simultaneous addition of a negatively charged siloxane reagent and a positive silicate at the same time as the addition of the positive silicate can effectively increase the surface charge between the nanoprobes and increase the electrostatic repulsion between the nanoparticles, which is conducive to the formation of monodisperse nanoprobes. The method described in the present application can successfully prepare high-performance oxygen-sensitive fluorescent nanoprobes with high brightness, good light stability, 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 positive silicate, and the mixture D is 1:(5-20):(30-100).

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

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

[0025] The positively charged silane reagent includes but is not limited to 3-aminopropyl trimethoxysilane, 3-aminopropyl triethoxysilane, 3-aminobutyl triethoxysilane, etc.

[0026] According to the specific embodiment of the present application, the protective colloid is polyvinylpyrrolidone. 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 the nanoparticles, thereby helping to control the particle size and dispersibility of the nanoparticles. Preferably, the viscosity K value of polyvinylpyrrolidone in water is between 12 and 120, so as to further optimize the particle size and dispersibility of the nanoprobe.

[0027] The negatively charged siloxane reagent includes, but is not limited to, 3-(trihydroxysilyl)propyl methyl phosphonic acid sodium salt, 3-(trihydroxysilyl)-propane sulfonic acid, triethoxysilyl propyl maleic acid, etc.

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

[0029] Optionally, the preparation method of the nanoprobe further comprises step 7): modifying the surface of the nanoparticles obtained after step 6) with other fluorescent probes or organelle or subcellular organelle targeting groups. By modifying the surface of the nanoprobe with cell or subcellular organelle targeting groups, the nanoprobe can be accurately positioned on the cell or subcellular organelle, realizing real-time and quantitative measurement of oxygen concentration in living cells. By using inhibitors to regulate mitochondrial function, the oxygen-sensitive probe claimed in the present application is successfully used for determination of oxygen consumption rate OCR in single living cells and drawing of cell energy metabolism map.

[0030] The present application also provides an oxygen-sensitive fluorescent nanoprobe, which has obvious core-shell structure characteristics, with a saturated fatty acid hydrophobic core embedding a hydrophobic oxygen-sensitive dye in the inside, and a hydrophilic silica shell layer in the outside. The silica material has good gas permeability and excellent biocompatibility, and by embedding the oxygen-sensitive probe in the inside of the nanoparticle, 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 light stability, good surface modifiability and biocompatibility; combined with fluorescence imaging technology, the energy metabolism analysis of single living cells can be realized by using the nanoprobe.

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

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

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

[0034] 1. The oxygen-sensitive fluorescent nanoprobes prepared by the method have the advantages that hydrophobic oxygen-sensitive dyes and hydrophilic silica materials with incompatible chemical properties are compatible in the same nanoparticle; the nanoprobes have a unique core-shell structure, excellent light stability, good oxygen response sensitivity and reversibility, good biocompatibility and surface modifiability, and in combination with fluorescence imaging technology, the nanoprobes can realize real-time measurement of intracellular oxygen concentration of a single living cell and be used for analyzing energy metabolism state of the cell.

[0035] 2. The hydrophobic oxygen-sensitive dyes dissolved in saturated fatty acids are located at the core part of the nanoprobes, so that the dyes are prevented from being disturbed by external environment, have extremely high light stability, and improve the detection accuracy; meanwhile, the design of the core / shell structure also avoids aggregation quenching and leakage of the oxygen-sensitive dyes.

[0036] 3. The fatty acids are externally grown with a silica shell layer, so that the structure of the entire material is reinforced, and the nanoprobes are also endowed with good biocompatibility, and other fluorescent probes or organelle targeting groups can be modified on the surface of the nanoprobes, which is beneficial to further functionalization of the nanoprobes.

[0037] 4. When the oxygen-sensitive fluorescent nanoprobes are exposed to different oxygen concentration atmospheres and irradiated with suitable excitation light, the fluorescence intensity and fluorescence lifetime of the nanoprobes will change significantly, and the nanoprobes can be used for long-time continuous and accurate monitoring of intracellular oxygen concentration, and in combination with fluorescence imaging technology, the energy metabolism state of a single living cell can be measured in real time. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 . Transmission electron microscope image of the oxygen-sensitive fluorescent nanoprobes prepared according to Embodiment 1.

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

[0040] Figure 3 . Light stability test curve of the oxygen-sensitive fluorescent nanoprobes prepared according to Embodiment 1.

[0041] Figure 4 . Curve of change of fluorescence intensity of the oxygen-sensitive fluorescent nanoprobes prepared according to Embodiment 1 with time under anaerobic-air atmosphere cycle switching.

[0042] Figure 5The oxygen-sensitive fluorescent nanoprobe prepared according to Example 1 (blue bar) measured the oxygen consumption rate of single PC12 cells treated with a mitochondrial inhibitor, and the Agilent Seahorse XFe96 cell energy analyzer (red bar) measured the oxygen consumption rate of PC12 cells treated with a mitochondrial inhibitor (cell number: 20000). DETAILED DESCRIPTION

[0043] The application is further illustrated by the following examples.

[0044] Example 1

[0045] 1) 0.84 mg of platinum (II) meso-tetra (pentafluorophenyl) porphine was weighed and dissolved in 1.0 mL of liquid stearic acid to form a red transparent mixture A at 70°C under stirring; the mass ratio of dye to stearic acid was 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 to form a uniform emulsion B; the volume ratio of mixture A to ultrapure water was 1:200;

[0047] 3) 134 μL of 3-aminopropyltrimethoxysilane was added to emulsion B, and a transparent

[0048] pink microemulsion C was formed under stirring; the volume ratio of 3-aminopropyltrimethoxysilane to emulsion B was 1:150;

[0049] 4) 200.0 mg of polyvinylpyrrolidone K-90 was added to microemulsion C to stabilize microemulsion C, and mixture D was obtained; the mass ratio of polyvinylpyrrolidone K-90 to microemulsion C was 1:100;

[0050] 5) 200 μL of 3-(trihydroxysilyl)propyl methyl phosphonic acid sodium salt and 1.0 mL of

[0051] tetraethyl orthosilicate were added to mixture D to obtain mixture E; the volume ratio of 3-(trihydroxysilyl)propyl methyl phosphonic acid sodium salt, tetraethyl orthosilicate, and mixture D was 1:5:100;

[0052] 6) Mixture E was continuously stirred at 70°C for 48 hours;

[0053] 7) The formed nanoparticle suspension was cooled to room temperature, and centrifuged with ultrapure water at a speed of 9000 g for 3 times to obtain an oxygen-sensitive fluorescent nanoprobe.

[0054]

[0055] Figure 1 ​The transmission electron microscope image of the oxygen-sensitive fluorescent nanoprobes prepared above shows that the prepared fluorescent nanoprobes have small nanoparticle size, and the average nanoparticle size is 174 nm.

[0056] Example 2

[0057] 1) 26.49 mg of tetraphenyl-palladium tetraphenylporphyrin complex was weighed and dissolved in 1.0 mL of liquid lauric acid to form a green transparent mixture A under stirring at 45°C; the mass ratio of the dye to lauric acid was 3.0:100;

[0058] 2) Then, 400 μL of the 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 the mixture A to ultrapure water was 1:50;

[0059] 3) 100.5 μL of 3-aminopropyltriethoxysilane was added to the emulsion B under stirring to form a transparent green microemulsion C; the volume ratio of 3-aminopropyltriethoxysilane to the emulsion B was 1:200;

[0060] 4) 800.0 mg of polyvinylpyrrolidone K-60 was added to the microemulsion C to stabilize the microemulsion C, and a mixture D was obtained; the mass ratio of polyvinylpyrrolidone K-60 to the microemulsion C was 1:25;

[0061] 5) 400 μL of 3-(trihydroxysilyl)-propane sulfonic acid and 2.0 mL of tetramethoxysilane were added to the mixture D to obtain a mixture E; the volume ratio of 3-(trihydroxysilyl)-propane sulfonic acid, tetramethoxysilane, and the mixture D was 1:5:50;

[0062] 6) The mixture E was continuously stirred at 60°C for 36 hours;

[0063] 7) The formed nanoparticle suspension was cooled to room temperature, and centrifuged with ultrapure water at a speed of 9000 g for 3 times to obtain the oxygen-sensitive fluorescent nanoprobes.

[0064] Example 3

[0065] 1) 0.906 mg of platinum octaethylporphyrin was weighed and dissolved in 1.0 mL of nonanoic acid to form a red transparent mixture A under stirring at room temperature; the mass ratio of the dye to nonanoic acid was 1.0:100;

[0066] 2) Then, 200 μL of the 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 the mixture A to ultrapure water was 1:100;

[0067] 3) 100.5 μL of 3-aminopropyltriethoxysilane was added to the emulsion B under stirring to form a transparent green microemulsion C; the volume ratio of 3-aminopropyltriethoxysilane to the emulsion B was 1:200;

[0068] 3) Add 202.0 μL of 3-aminobutyltriethoxysilane to emulsion B, a transparent pink microemulsion C can be formed under stirring;

[0069] The volume ratio of 3-aminobutyltriethoxysilane to emulsion B is 1:100;

[0070] 4) Add 2000.0 mg of polyvinylpyrrolidone K-30 to microemulsion C to stabilize microemulsion C, obtaining mixture D; the mass ratio of polyvinylpyrrolidone K-30 to microemulsion C is 1:10;

[0071] 5) Add 666.6 μL of triethoxysilylpropylmaleic acid and 3.3 mL of tetraethyl orthosilicate to mixture D, obtaining mixture E; the volume ratio of triethoxysilylpropylmaleic acid, tetraethyl orthosilicate, and mixture D is 1:5:30;

[0072] 6) Continue to stir mixture E at 45°C for 24 hours;

[0073] 7) Cool the formed nanoparticle suspension to room temperature, and centrifuge and wash with ultrapure water at 9000g for 3 times to obtain the oxygen-sensitive fluorescent nanoprobe.

[0074] Test 1 Analysis of the fluorescence intensity responsiveness of the nanoprobe under different oxygen concentration gas atmospheres

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

[0076] 2) Adjust the gas flow meters to keep the total flow rate of nitrogen and oxygen constant, and prepare different oxygen concentration gas atmospheres 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 a stabilization period;

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

[0079] 5) According to the Stern-Volmer equation, calculate the I / I value under each oxygen concentration, where I represents the fluorescence intensity under nitrogen-saturated gas atmosphere, and I represents the fluorescence intensity under the current oxygen concentration. Plot the Stern-Volmer curve with I / I as the vertical coordinate and the oxygen content volume percentage as the horizontal coordinate.

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

[0081] Photostability evaluation of the experimental 2-nanometer probe

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

[0083] 2) Take 200 μL of the mixture and drop it onto the surface of the PET plastic film;

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

[0085] 4) Place the dried membrane in a cuvette and test the photostability of the oxygen-sensitive fluorescent nanoprobe in air using a fluorescence spectrometer. The excitation wavelength is 390 nm, the emission wavelength is 650 nm, the excitation slit width is 20 nm, and the emission slit width is 5 nm.

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

[0087] Verification of the reversibility of the oxygen response of the experimental 3-nanometer probe

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

[0089] 2) Take 200 μL of the mixture and drop it onto the surface of the PET plastic film;

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

[0091] 4) Place the dried membrane in a cuvette and use a fluorescence spectrometer to test the oxygen response reversibility of the oxygen-sensitive fluorescent nanoprobe in an oxygen-free-air atmosphere. The excitation wavelength is 390 nm and the emission wavelength is 650 nm.

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

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

[0094] Experiment 4 used nanoprobes to detect intracellular oxygen concentration and analyze energy metabolism status.

[0095] 1) PC12 cells (2 x 10 5 cells / well) were seeded into 24-well plates and incubated in DMEM medium for 24 hours;

[0096]

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

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

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

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

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

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

[0103] 3) The inhibitors were dissolved in DMSO to form an inhibitor-containing DMSO solution with an inhibitor concentration of 1.0 mM;

[0104] 4) 50.0 μL of the inhibitor-containing DMSO solution was mixed with 9.95 mL of DMEM medium to form a DMEM medium containing 5.0 μM of the inhibitor;

[0105] 5) 200 μL of the DMEM medium containing 5.0 μM of the inhibitor was added to the 24-well plate, and 500 μL of mineral oil was added above the medium to form a gas barrier layer;

[0106] 6) The luminescence lifetime of the oxygen-sensitive fluorescent nanoprobes was recorded using a fluorescence lifetime imaging microscope for 180

[0107] minutes, with a measurement time interval of 10 minutes.

[0108] The change trend of the oxygen concentration in PC12 cells was calculated according to the standard working curve of the oxygen-sensitive fluorescent nanoprobes (control group) and the change in their luminescence lifetime (when using inhibitors), and then the amount of oxygen consumed in the first minute was calculated; ​

[0109] Cellular energy metabolism analysis experiments were performed according to the instructions of Seahorse XFe96 Cell Energy Metabolic Analyzer and the data were compared with the data of the present application.

[0110] The above-mentioned inhibitor is a mitochondrial inhibitor, and in the present test, it is specifically rotenone.

[0111] Figure 5 The oxygen consumption rate profile of single PC12 cells treated with mitochondrial inhibitor was measured for the oxygen-sensitive fluorescent nanoprobes prepared according to Example 1 (blue bar) and the oxygen consumption rate profile of PC12 cells treated with mitochondrial inhibitor (cell number: 20000) was measured by Agilent Seahorse XFe96 Cell Energy Analyzer (red bar). By regulating mitochondrial function using the inhibitor, the oxygen-sensitive probes claimed in the present application were successfully used for the determination of oxygen consumption rate OCR in single living cells.

Claims

1. A method for preparing an oxygen-sensitive fluorescent nanoprobe, characterized in that, Includes the following steps: 1) Dissolve the hydrophobic oxygen-sensitive dye in saturated fatty acid to form mixture A; 2) Mix mixture A with ultrapure water and stir vigorously until a homogeneous emulsion B is formed; 3) Mix the positively charged siloxane reagent with emulsion B to form a transparent microemulsion C under stirring; 4) Add a protective colloid to the microemulsion C to obtain a stabilized mixture D; wherein the protective colloid has the function of preventing collisions and agglomeration between nanoparticles and promoting uniform dispersion of nanoparticles. 5) Mix the negatively charged siloxane reagent, orthosilicate, and mixture D to obtain mixture E; 6) Mixture E is continuously stirred and reacted at 20-100℃ to form a silica shell. After the reaction is completed, the product is centrifuged and washed. The protective colloid is polyvinylpyrrolidone; The hydrophobic oxygen-sensitive dye is selected from one of the oxygen-sensitive probes composed of porphyrin or porphyrin complexes of platinum, palladium, ruthenium, and iridium. The positively charged silane reagent is selected from one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, and 3-aminobutyltriethoxysilane; The negatively charged siloxane reagent is selected from one of the following: sodium 3-(trihydroxysilyl)propylmethylphosphonate, 3-(trihydroxysilyl)-propanesulfonic acid, and triethoxysilylpropylmaleic acid.

2. The preparation method according to claim 1, characterized in that, The carbon chain of the saturated fatty acid has 6 to 20 carbon atoms.

3. The preparation method according to claim 1, characterized in that, In step 1), the mass ratio of the hydrophobic oxygen-sensitive dye to saturated fatty acid is (0.1-3):100; in step 2), the volume ratio of 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 microemulsion C is 1:(10-100); in step 5), the volume ratio of the negatively charged siloxane reagent, orthosilicate, and mixture D is 1:(5-20):(30-100).

4. The application of the oxygen-sensitive fluorescent nanoprobe prepared by the preparation method according to any one of claims 1-3 in the detection of oxygen concentration in living cells and the analysis of energy metabolism in living cells.

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