CN113416538A - Transition metal oxide nano probe, preparation method and application - Google Patents
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Abstract
Discloses a transition metal oxide nano probe, a preparation method and application, has low cost, can carry out fluorescence imaging on specific protein, and can be used for simultaneously imaging a plurality of proteins. The transition metal oxide nanoprobe includes: the nano-particles comprise transition metal oxide nano-particles and modified molecules compounded on the surfaces of the transition metal oxide nano-particles, wherein the transition metal oxide comprises oxides or hydroxides of three elements of iron, cobalt and nickel, and the surface modified molecules comprise biological protein molecules, biotin molecules and fluorescein molecules.
Description
Technical Field
The invention relates to the technical field of nano materials and biological analysis, in particular to a transition metal oxide nano probe, a preparation method of the probe and application of the transition metal oxide nano probe in cell specific protein imaging analysis.
Background
The nano probe is a nano material based on self physicochemical properties and can be used for assisting in improving imaging quality of a certain medical imaging instrument. Most of the present nanoprobes for single-cell level protein molecule imaging are based on the principle of antibody-antigen immune effect, and can realize imaging of 5-8 markers at most by signal selection, but are only suitable for semi-quantitative analysis of high-abundance expressed proteins. The secondary antibody label based on the inorganic metal element label can realize the expression analysis of dozens of protein molecules, and the quantitative analysis method based on the inorganic mass spectrum has high quantitative sensitivity, but the destructive quantitative process causes the loss of the spatial distribution information of the protein. Meanwhile, although signal amplification and imaging can be realized by adopting the secondary antibody to identify the primary antibody, the secondary antibody has limited carrying capacity for the metal label, is relatively expensive and has species selectivity of the primary antibody. Therefore, designing a low-cost nanoprobe that can achieve broad-spectrum imaging of specific protein targets at single-cell level is one of the hot researches widely performed by researchers in the field at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a transition metal oxide nanoprobe and a preparation method thereof, which have low cost, can perform fluorescence imaging on specific protein and can be used for simultaneously imaging a plurality of proteins.
The technical scheme of the invention is as follows: the transition metal oxide nanoprobe includes: the nano-particles comprise transition metal oxide nano-particles and modified molecules compounded on the surfaces of the transition metal oxide nano-particles, wherein the transition metal oxide comprises oxides or hydroxides of three elements of iron, cobalt and nickel, and the surface modified molecules comprise biological protein molecules, biotin molecules and fluorescein molecules.
The preparation method of the transition metal oxide nanoprobe comprises the following steps:
(1) dissolving a mixture of biotin, N-hydroxysuccinimide NHS and 1-ethyl-3- (3' dimethylaminopropyl) carbodiimide EDC in 2- (N-morpholine) ethanesulfonic acid MES buffer solution, and violently stirring for a period of time at room temperature in a dark condition to obtain an activated biotin solution; then carrying out high-speed centrifugal purification to obtain activated biotin, and re-dissolving the activated biotin in dimethyl sulfoxide (DMSO) to obtain a first solution;
(2) dissolving 5-50 g/L of a biological protein molecular solution in a sodium bicarbonate buffer solution, and uniformly mixing to obtain a second solution;
(3) uniformly mixing the first solution and the second solution, reacting for a period of time to obtain biotin protein, and then dialyzing to obtain a purified biotin protein solution;
(4) mixing and reacting a solution containing transition metal elements and a biotin protein solution for a period of time, adding a sodium hydroxide aqueous solution to adjust the pH value to be more than 10, continuously and violently stirring for a period of time at a constant temperature of 37 ℃, standing for a period of time and aging to obtain a transition metal oxide nanoparticle solution, and dialyzing and purifying;
(5) adding a sodium bicarbonate buffer solution with the pH value of 9 into the transition metal oxide nanoparticle solution obtained in the step (4), and placing the solution in an environment with the temperature of 4 ℃ and stirring to reduce the temperature to be close to 4 ℃; the final concentration of the sodium bicarbonate buffer solution is 0.5-10 mg/ml calculated by the protein molecular concentration;
(6) slowly dripping RBITC or FITC dimethylformamide DMF or dimethyl sulfoxide DMSO solution with a certain concentration into the reaction solution in the step (5); rapidly injecting an ammonium chloride solution with a certain concentration to terminate the coupling reaction; and after continuously stirring for 1-5 hours, stopping stirring, and performing dialysis purification treatment to obtain the final transition metal oxide nano probe.
Compared with the nano probe with the antibody as the modified molecule, the invention uses the cheaper biotin to modify the surface of the nano particle based on the characteristic of the specific binding between the biotin and the streptavidin, thereby greatly reducing the preparation cost of the probe, getting rid of the constraint that the antibody only specifically binds to the specific protein, simultaneously realizing the common imaging of various proteins, combining the content of the transition metal element in the probe, and quantitatively analyzing the expression quantity of the specific protein in the cell.
The transition metal oxide nano probe is also provided to be applied to a fluorescence imaging experiment, and the method comprises the following steps: when the cell specific protein is subjected to imaging analysis, the selected cell line is digested, planted on a cover glass and fixed, and then the cells are respectively subjected to sealing treatment by using serum albumin and a biotin sealing kit; then carrying out primary antibody incubation for 12 hours with a commercial biotinylated antibody of a specific protein; after incubation, the treated cells were washed three times with PBS and incubated with ChromeoTM546 streptavidin SC-364714 at 37 degrees C environment continued to incubate for 1 hours; and (4) washing with PBS three times, continuously incubating the cells with the nano probe solution obtained in the step (6) for a period of time in an environment at 37 ℃, and performing a fluorescence imaging experiment after washing with PBS three times.
Drawings
FIG. 1 is a schematic diagram of the synthesis and functionalization of a series of functional fluorescent nanoprobes prepared according to the present invention;
FIG. 2 is a graph of dynamic light scattering characterization of a series of transition metal oxide nanoprobes prepared in examples 1, 2, 3 and 4 of the present invention;
FIG. 3 is a graph of fluorescence imaging of HER-2 protein using the iron oxide nanoprobe prepared in example 1 of the present invention in example 5;
FIG. 4 is a graph of simultaneous fluorescence imaging of HER-2 protein and Ki67 protein using the cobalt hydroxide nanoprobe prepared in example 2 of the invention and the ferroferric oxide nanoprobe prepared in example 4 of the invention as described in example 6 of the invention.
Fig. 5 is a flowchart of a method for preparing a transition metal oxide nanoprobe according to the present invention.
Detailed Description
The transition metal oxide nanoprobe includes: the nano-particles comprise transition metal oxide nano-particles and modified molecules compounded on the surfaces of the transition metal oxide nano-particles, wherein the transition metal oxide comprises oxides or hydroxides of three elements of iron, cobalt and nickel, and the surface modified molecules comprise biological protein molecules, biotin molecules and fluorescein molecules.
Preferably, the hydrated particle size of the transition metal oxide nanoprobe is 10-50 nm.
Preferably, the molar ratio of the transition metal element in the transition metal oxide to the bioprotein molecule is 1: a, a is 0.005-0.1.
Preferably, the molar ratio of the bioprotein molecules to the biotin molecules is 1: b, b is 5-100; the mol ratio of the biological protein molecules to the fluorescein molecules is 1: d, d is 1 to 10
Preferably, when the transition metal oxide nanoprobe is used for in situ imaging of a specific protein in a cell, the cell is subjected to antibody labeling treatment of the specific protein modified by biotin and streptavidin incubation treatment.
Preferably, the biological protein molecule is one or more of bovine serum albumin, human serum albumin, ferritin, fibronectin, egg white protein.
Preferably, the fluorescein molecule is rhodamine B isothiocyanate RBITC or fluorescein isothiocyanate FITC.
As shown in fig. 5, there is also provided a method for preparing the transition metal oxide nanoprobe, which comprises the following steps:
(1) dissolving a mixture of biotin, N-hydroxysuccinimide NHS and 1-ethyl-3- (3' dimethylaminopropyl) carbodiimide EDC in 2- (N-morpholine) ethanesulfonic acid MES buffer solution, and violently stirring for a period of time at room temperature in a dark condition to obtain an activated biotin solution; then carrying out high-speed centrifugal purification to obtain activated biotin, and re-dissolving the activated biotin in dimethyl sulfoxide (DMSO) to obtain a first solution;
(2) dissolving 5-50 g/L of a biological protein molecular solution in a sodium bicarbonate buffer solution, and uniformly mixing to obtain a second solution;
(3) uniformly mixing the first solution and the second solution, reacting for a period of time to obtain biotin protein, and then dialyzing to obtain a purified biotin protein solution;
(4) mixing and reacting a solution containing transition metal elements and a biotin protein solution for a period of time, adding a sodium hydroxide aqueous solution to adjust the pH value to be more than 10, continuously and violently stirring for a period of time at a constant temperature of 37 ℃, standing for a period of time and aging to obtain a transition metal oxide nanoparticle solution, and dialyzing and purifying;
(5) adding a sodium bicarbonate buffer solution with the pH value of 9 into the transition metal oxide nanoparticle solution obtained in the step (4), and placing the solution in an environment with the temperature of 4 ℃ and stirring to reduce the temperature to be close to 4 ℃; the final concentration of the sodium bicarbonate buffer solution is 0.5-10 mg/ml calculated by the protein molecular concentration; the preferable concentration is 1-5 mg/ml, and the more preferable concentration is 2-4 mg/ml;
(6) slowly dripping RBITC or FITC dimethylformamide DMF or dimethyl sulfoxide DMSO solution with a certain concentration into the reaction solution in the step (5); rapidly injecting an ammonium chloride solution with a certain concentration to terminate the coupling reaction; and after continuously stirring for 1-5 hours, stopping stirring, and performing dialysis purification treatment to obtain the final transition metal oxide nano probe.
Compared with the nano probe with the antibody as the modified molecule, the invention uses the cheaper biotin to modify the surface of the nano particle based on the characteristic of the specific binding between the biotin and the streptavidin, thereby greatly reducing the preparation cost of the probe, getting rid of the constraint that the antibody only specifically binds to the specific protein, simultaneously realizing the common imaging of various proteins, combining the content of the transition metal element in the probe, and quantitatively analyzing the expression quantity of the specific protein in the cell.
Preferably, in the step (1), the feeding molar ratio of biotin to EDC to NHS is 1: e: 1, e is 1-1.2, the mixing reaction time of biotin, EDC and NHS is 0.5-12 hours, and the centrifugal purification condition is that the mixture is centrifuged for 5-30 minutes at 8000-14000 rpm;
in the step (3), the ratio of the biological protein molecules to the activated biological protein molecules is 1: b, b is 5-100, the reaction condition is light-proof and violent stirring, the reaction time is 2-24 hours, the dialysis condition is that a dialysis bag with the cut-off molecular weight of 3000-15000 is selected, and the dialysis time is 1-3 days;
in the step (4), the transition metal element solution is a solution containing transition metal ions with a certain concentration, which is obtained by dissolving inorganic salts or acetates of iron, cobalt and nickel such as chlorides, sulfates and nitrates in water, wherein the concentration of the transition metal ions is 0.05-1 mol/l, and the ratio of biotin protein molecules to the transition metal elements is 1: c, c is 10-200, stirring and reacting for 1-10 hours after adding the sodium hydroxide aqueous solution, standing and aging for 8-24 hours, and dialyzing the purified transition metal oxide solution under the condition that a dialysis bag with the cut-off molecular weight of 3000-15000 is selected for 1-3 days;
in the step (6), the concentration of DMF or DMSO solution of RBITC or FITC is 2-20 g/L, the molar ratio of RBITC or FITC to protein molecules in the transition metal oxide reaction solution obtained in the step (5) is 1: f, f is 0.1-1, the reaction is completed in a low-temperature environment at 4 ℃, the concentration of ammonium chloride solution is 1-10 mol/L, a dialysis bag with the molecular weight cutoff of 3000-15000 is selected during purification, and the dialysis time is 1-3 days.
The transition metal oxide nano probe is also provided to be applied to a fluorescence imaging experiment, and the method comprises the following steps: when the cell specific protein is subjected to imaging analysis, the selected cell line is digested, planted on a cover glass and fixed, and then the cells are respectively subjected to sealing treatment by using serum albumin and a biotin sealing kit; then carrying out primary antibody incubation for 12 hours with a commercial biotinylated antibody of a specific protein; after incubation, the treated cells were washed three times with PBS and incubated with ChromeoTM546 streptavidin SC-364714(Santa Cruz Biotechnology, SC-364714) was incubated for an additional 1 hour at 37 degrees Celsius; and (4) washing with PBS three times, continuously incubating the cells with the nano probe solution obtained in the step (6) for a period of time in an environment at 37 ℃, and performing a fluorescence imaging experiment after washing with PBS three times.
The preferred concentration of the cells incubated with the biotinylated antibody depends on the cell line and the type of protein, and the ratio of the number of cells to the number of antibody molecules is generally 1: g, g is 10 to 200.
Preferably, streptavidin and biotinylated antibody are used in a molar ratio of 1: h, h is 0.3-0.8.
The preferable incubation time of the nanoprobe solution and the treated cells in the environment of 37 ℃ is 15 minutes to 4 hours, and the more preferable incubation time is 0.5 to 1 hour.
The concentration of the nano-probe solution which is incubated with the treated cells in the environment of 37 ℃ is preferably 2-50 mg/L calculated by protein molecules, and the concentration of the solution is more preferably 5-20 mg/L.
Experimental results show that the constructed series of nano probes can specifically bind to related proteins, realize in-situ imaging of cells, and can be used for simultaneous in-situ imaging of two or more proteins.
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
Preparation of iron oxide Probe
A) Biotin, NHS and EDC were mixed in MES buffer solution in a molar ratio of 1:1:1, and vigorously stirred at room temperature for 30 minutes in the absence of light to obtain an activated biotin solution. Subsequently, the reaction mixture was centrifuged at 9000 rpm for 5 minutes in a high-speed centrifuge, and after the supernatant was discarded, the precipitate was redissolved in DMSO to obtain reaction mixture 1.
B) Bovine serum albumin (hereinafter referred to as BSA) was added to a final concentration of 10 g/l in a sodium bicarbonate buffer solution of 0.1 mol/l and pH 9, and recorded as reaction solution 2;
C) and B, uniformly mixing the solution 1 obtained in the step A and the solution 2 obtained in the step B, wherein the ratio of BSA molecules to activated biotin molecules is 1: and 10, reacting for 3 hours in the dark, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the purified biotin protein.
D) Weighing anhydrous ferric chloride and ferrous chloride hexahydrate as Fe2+:Fe3+The molar ratio of 2:1 was dissolved in water so that the final total iron element molar concentration was 0.2 moles per liter.
E) And C, mixing 0.6 ml of the ferric chloride solution obtained in the step D with the biotin protein solution (6 ml, 20 g per liter) obtained in the step C for reaction for 5 minutes, adding 0.3 ml of sodium hydroxide solution with the concentration of 1 mol per liter, continuously and violently stirring for 12 hours at a constant temperature of 37 ℃, and dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000 to obtain the iron oxide nanoparticle probe.
F) And E, adding 0.1 mol/L sodium bicarbonate buffer solution with the pH of 9 into the iron oxide solution finally obtained in the step E, wherein the final concentration is 3.6 milligrams per milliliter calculated by the protein molecule concentration, and placing the solution in an environment with the temperature of 4 ℃ to stir so that the temperature of the solution is reduced to be close to 4 ℃.
G) 10 grams per liter of the DMF solution of RBITC was slowly added dropwise to the reaction solution of step F above, such that the final molar ratio of RBITC to protein molecules was 2: 1. Finally, a 4.6 molar solution of ammonium chloride was rapidly injected to terminate the coupling reaction. And after stirring for 2 hours, stopping stirring, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the final iron oxide nano probe.
Example 2
Preparation of cobalt hydroxide Probe
A) Adding 2 ml of cobalt nitrate solution with the concentration of 50 mmol per ml into 10 ml of fibronectin solution with the concentration of 20 mg per ml, stirring for 5 minutes to mix uniformly, adding 0.2 ml of sodium hydroxide solution with the concentration of 2 mol per liter to raise the final pH to more than 10, stirring vigorously for 2 hours, standing for 12 hours for aging, dialyzing for 36 hours, and freeze-drying to obtain the fibronectin mineralized cobalt hydroxide nanoparticles.
B) Biotin, NHS and EDC were mixed in MES buffer solution in a molar ratio of 1:1:1.2, and vigorously stirred at room temperature for 50 minutes in the absence of light to obtain an activated biotin solution. The pellet was then centrifuged at 7000 rpm for 5 minutes in a high speed centrifuge, and after discarding the supernatant, the pellet was redissolved in DMSO.
C) The fibronectin mineralized cobalt hydroxide nanoparticles from step a were added to 0.1 mol/l sodium bicarbonate buffer pH 9 at a final fibronectin concentration of 5 grams/l and the solution from step B was mixed such that the ratio of fibronectin to activated biotin molecules was 1: and 20, keeping away from light for 3 hours, dialyzing for 2 days by using a dialysis bag with the cut-off molecular weight of 13000, and then freeze-drying to obtain the purified biotin-modified cobalt fibronectin hydroxide nanoprobe.
D) And E, adding 0.1 mol/L sodium bicarbonate buffer solution with the pH of 9 into the solution prepared by the biotin-modified fibronectin cobalt hydroxide nanoprobe finally obtained in the step E, calculating the final concentration to be 2 mg/ml according to the protein molecule concentration, and placing the solution in an environment with the temperature of 4 ℃ and stirring to reduce the temperature to be close to 4 ℃.
E) 10 grams per liter of the DMF solution of RBITC was slowly added dropwise to the reaction solution of step F above, such that the final molar ratio of RBITC to protein molecules was 4: 1. Finally, a 4.6 molar solution of ammonium chloride was rapidly injected to terminate the coupling reaction. And after stirring for 2 hours, stopping stirring, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the final cobalt hydroxide nano probe.
Example 3
Preparation of Nickel oxide Probe
A) Biotin, NHS and EDC were mixed in MES buffer solution in a molar ratio of 1:1:1.1, and vigorously stirred at room temperature in the dark for 30 minutes to obtain an activated biotin solution. Then, the mixture was centrifuged at 10000 rpm in a high speed centrifuge for 10 minutes, and after the supernatant was discarded, the precipitate was redissolved in DMSO to obtain reaction solution 1.
B) Human serum albumin (hereinafter referred to as HSA) was added to a sodium bicarbonate buffer solution of 0.1 mol/l and pH 9 at a final concentration of 15 g/l, and this was designated as reaction solution 2;
C) and B, uniformly mixing the solution 1 obtained in the step A and the solution 2 obtained in the step B, wherein the ratio of BSA molecules to activated biotin molecules is 1: and 10, reacting for 3 hours in the dark, dialyzing for 1 day by using a dialysis bag with the molecular weight cutoff of 8000, and freeze-drying to obtain the purified biotin protein.
D) Nickel chloride was weighed and dissolved in water so that the molar concentration of the finally obtained nickel element was 0.1 mol per liter.
E) And (3) mixing 1.2 ml of the nickel chloride solution obtained in the step (D) with the biotin protein solution (6 ml, 20 g per liter) obtained in the step (C) for reaction for 5 minutes, adding 0.5 ml of sodium hydroxide solution with the concentration of 1 mol per liter, continuously and violently stirring at the constant temperature of 37 ℃ for 12 hours, and dialyzing for 2 days by using a dialysis bag with the molecular weight cutoff of 15000 to obtain the nickel oxide nanoparticle probe.
F) And E, adding 0.1 mol/L sodium bicarbonate buffer solution with the pH of 9 into the iron oxide solution finally obtained in the step E, wherein the final concentration is 5 mg/ml calculated by the protein molecule concentration, and placing the solution in an environment with the temperature of 4 ℃ and stirring to reduce the temperature to be close to 4 ℃.
G) 10 grams per liter of the DMF solution of RBITC was slowly added dropwise to the reaction solution of step F above, such that the final molar ratio of RBITC to protein molecules was 3: 1. Finally, a 4.6 molar solution of ammonium chloride was rapidly injected to terminate the coupling reaction. And after stirring for 2 hours, stopping stirring, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the final nickel oxide nano probe.
Example 4
A) To 10 ml of bovine serum albumin solution at a concentration of 50 mg per ml was added 5 ml of ferric chloride to ferrous chloride at a total iron concentration of 50 mmol per ml of 2:1, stirring for 5 minutes to uniformly mix, adding 0.2 ml of 1 mol/L sodium hydroxide solution to raise the final pH to above 10, stirring vigorously for 12 hours, dialyzing for 36 hours, and freeze-drying to obtain bovine serum albumin mineralized ferroferric oxide nanoparticles.
B) Biotin, NHS and EDC were mixed in MES buffer solution in a molar ratio of 1:1:1.2, and vigorously stirred at room temperature for 50 minutes in the absence of light to obtain an activated biotin solution. The pellet was then centrifuged at 7000 rpm for 5 minutes in a high speed centrifuge, and after discarding the supernatant, the pellet was redissolved in DMSO.
C) Adding the protein mineralized ferroferric oxide nanoparticles obtained in the step A into sodium bicarbonate buffer solution with the pH of 9 and the concentration of 5 grams per liter of final protein concentration, wherein the sodium bicarbonate buffer solution is 0.1 mole per liter of final protein concentration, and mixing the solution obtained in the step B to ensure that the ratio of the protein to the activated biotin molecules is 1: and 15, reacting for 3 hours in a dark place, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the purified biotin-modified bovine serum albumin ferroferric oxide nano probe.
D) And E, adding 0.1 mol/L sodium bicarbonate buffer solution with the pH of 9 into the solution prepared by the biotin-modified bovine serum albumin ferroferric oxide nanoprobe finally obtained in the step E, calculating the final concentration by the protein molecule concentration to be 3 mg/ml, and placing the solution in an environment with the temperature of 4 ℃ to stir so that the temperature of the solution is reduced to be close to 4 ℃.
E) 10 g/l of FITC in DMF was slowly added dropwise to the reaction solution from step F above, so that the final FITC to protein molecule molar ratio was 4: 1. Finally, a 4.6 molar solution of ammonium chloride was rapidly injected to terminate the coupling reaction. And after stirring for 2 hours, stopping stirring, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the final FITC modified iron oxide nano probe.
Referring to fig. 2, fig. 2 is a graph showing the dynamic light scattering characterization of a series of transition metal oxide nanoprobes prepared in examples 1, 2, 3 and 4 of the present invention, in which a is a graph showing the dynamic light scattering characterization of nanoprobes prepared in example 1 of the present invention, B is a graph showing the dynamic light scattering characterization of nanoprobes prepared in example 2 of the present invention, C is a graph showing the dynamic light scattering characterization of nanoprobes prepared in example 3 of the present invention, and D is a graph showing the dynamic light scattering characterization of nanoprobes prepared in example 4 of the present invention. As can be seen from FIG. 2, the transition metal oxide nanoprobes prepared according to the present invention have an average hydrated particle size of 10 to 50 nm.
Example 5
Cell specific protein imaging
A) After trypsinizing the mammary adenocarcinoma SK-BR-3 cell line, the seeds were incubated on coverslips for 12 hours at 37 ℃, the medium was removed and the cells were washed three times with PBS and fixed with 4% paraformaldehyde solution for 20 minutes at room temperature.
B) Cells were blocked with 3% Bovine Serum Albumin (BSA) solution in PBS buffer for 1 hour, then intracellular biotin was blocked with endogenous biotin blocking kit, and finally slides were washed 3 times with PBS buffer.
C) Respectively mixing the cell-loaded slide glass with a biotin-modified human epidermal growth factor receptor-2 (hereinafter, written as HER2) protein HER2-bio antibody (Thermo Fisher, MA5-12995) according to the ratio of the number of cells to the number of antibody moles of 1: 100 were incubated in a refrigerator at 4 ℃ for 12 hours and then washed three times with PBS buffer.
D) Mixing Chromeo TM546 streptavidin (Santa Cruz Biotechnology, SC-364714) was diluted 150-fold and incubated with the cell-loaded slides for 1 hour at 37 degrees celsius, with a molar ratio of streptavidin to biotinylated antibody of 1: 0.5. washed 3 times with PBS in the dark for 5 minutes each.
E) The cells were further incubated with the iron oxide nanoprobes obtained in example 1 at 37 ℃ for 50 minutes in E solution with a concentration of 10 mg per liter, washed three times with PBS, and then subjected to fluorescence imaging.
Experimental results show that the iron oxide nanoprobe constructed in the embodiment 1 can be specifically combined with HER-2 protein expressed by cells and realize single cell in-situ imaging.
Referring to fig. 3, fig. 3 is a graph showing HER-2 protein fluorescence imaging of the iron oxide nanoprobe prepared in example 1 of the present invention. As can be seen from FIG. 3, the iron oxide nanoprobe prepared in example 1 of the present invention can be used for imaging single cell scale HER-2 protein fluorescence, and clearly shows that HER-2 protein is mainly distributed on cell membrane of cell.
Example 6
Co-imaging of two specific proteins expressed in three cells
A) Three different types of human breast adenocarcinoma SK-BR-3, MCF-7, MDA-MB-231 cell lines were trypsinized, seeded on a cover glass, incubated at 37 ℃ for 12 hours, washed with PBS and fixed at room temperature for 20 minutes.
B) The cells were blocked with 3% BSAPBS solution for 1 hour, then the intracellular biotin was blocked with endogenous biotin blocking kit, and finally the slides were washed 3 times with PBS buffer.
C) Respectively mixing the cell-loaded slide glass with HER2 protein HER2-bio antibody (Thermo Fisher, MA5-12995) according to the ratio of cell number to antibody mole number of 1: 100 parts, or the biotin-modified relevant antigen of proliferating cells (Ki67-bio associated antibody) was incubated at 4 ℃ in a refrigerator at a ratio of 1:20 cell number to mole number of antibody (Novus, NB500-170B) for 12 hours, followed by three washes with PBS buffer.
D) Mixing Chromeo TM546 streptavidin (Santa Cruz Biotechnology, SC-364714) was diluted 150-fold and incubated with the cell-loaded slides for 1 hour at 37 degrees celsius, with a molar ratio of streptavidin to biotinylated antibody of 1: 0.8. washed 3 times with PBS in the dark for 5 minutes each.
E) The cells were incubated with the iron oxide nanoprobes obtained in example 1 and the ferroferric oxide nanoprobes obtained in example 4 at 37 ℃ for 50 minutes in a solution with a concentration of 10 mg per liter, washed three times with PBS, stained with a nuclear dye (designated as DAPI) for 30 minutes, washed three times with PBS, and subjected to fluorescence imaging experiments.
Referring to fig. 4, fig. 4 is a graph showing simultaneous fluorescence imaging of HER-2 protein and Ki67 protein using the iron oxide nanoprobe prepared in example 1 of the present invention and the ferroferric oxide nanoprobe prepared in example 4 of the present invention. As can be seen from FIG. 4, the iron oxide nanoprobe prepared in example 1 of the present invention can specifically bind to HER-2 protein expressed by cells, and clearly shows that HER-2 protein is mainly distributed on cell membranes; the ferroferric oxide nano probe prepared in the embodiment 4 of the invention can be specifically combined with Ki67 protein expressed by cells, and the position of the ferroferric oxide nano probe is clearly superposed with DAPI (nuclear dye) of cell nucleus, which shows that the Ki67 protein mainly focuses on cell nucleus aggregation; the nano fluorescent probes constructed in the embodiment 1 and the embodiment 4 can be used simultaneously, and have no influence on the labeling effect.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.
Claims (10)
1. Transition metal oxide nanoprobes, characterized in that: it includes: the nano-particles comprise transition metal oxide nano-particles and modified molecules compounded on the surfaces of the transition metal oxide nano-particles, wherein the transition metal oxide comprises oxides or hydroxides of three elements of iron, cobalt and nickel, and the surface modified molecules comprise biological protein molecules, biotin molecules and fluorescein molecules.
2. The transition metal oxide nanoprobe of claim 1, wherein: the hydrated particle size of the transition metal oxide nanoprobe is 10-50 nanometers.
3. The transition metal oxide nanoprobe of claim 2, wherein: the molar ratio of the transition metal element in the transition metal oxide to the biological protein molecule is 1: a, a is 0.005-0.1.
4. The transition metal oxide nanoprobe of claim 3, wherein: the molar ratio of the biological protein molecules to the biotin molecules is 1: b, b is 5-100; the mol ratio of the biological protein molecules to the fluorescein molecules is 1: d and d are 1-10.
5. The transition metal oxide nanoprobe of claim 4, wherein: when the transition metal oxide nano probe is used for in-situ imaging of specific protein in cells, the cells are subjected to antibody labeling treatment of the specific protein modified by biotin, and streptavidin incubation treatment is carried out.
6. The transition metal oxide nanoprobe of claim 5, wherein: the biological protein molecule is one or more of bovine serum albumin, human serum albumin, ferroglobin, fibronectin and egg white protein.
7. The transition metal oxide nanoprobe of claim 6, wherein: the fluorescein molecule is rhodamine B isothiocyanate RBITC or fluorescein isothiocyanate FITC.
8. The method for preparing a transition metal oxide nanoprobe according to claim 1, wherein: which comprises the following steps:
(1) dissolving a mixture of biotin, N-hydroxysuccinimide NHS and 1-ethyl-3- (3' dimethylaminopropyl) carbodiimide EDC in 2- (N-morpholine) ethanesulfonic acid MES buffer solution, and violently stirring for a period of time at room temperature in a dark condition to obtain an activated biotin solution; then carrying out high-speed centrifugal purification to obtain activated biotin, and re-dissolving the activated biotin in dimethyl sulfoxide (DMSO) to obtain a first solution;
(2) dissolving 5-50 g/L of a biological protein molecular solution in a sodium bicarbonate buffer solution, and uniformly mixing to obtain a second solution;
(3) uniformly mixing the first solution and the second solution, reacting for a period of time to obtain biotin protein, and then dialyzing to obtain a purified biotin protein solution;
(4) mixing and reacting a solution containing transition metal elements and a biotin protein solution for a period of time, adding a sodium hydroxide aqueous solution to adjust the pH value to be more than 10, continuously and violently stirring for a period of time at a constant temperature of 37 ℃, standing for a period of time and aging to obtain a transition metal oxide nanoparticle solution, and dialyzing and purifying;
(5) adding a sodium bicarbonate buffer solution with the pH value of 9 into the transition metal oxide nanoparticle solution obtained in the step (4), and placing the solution in an environment with the temperature of 4 ℃ and stirring to reduce the temperature to be close to 4 ℃; the final concentration of the sodium bicarbonate buffer solution is 0.5-10 mg/ml calculated by the protein molecular concentration;
(6) slowly dripping RBITC or FITC dimethylformamide DMF or dimethyl sulfoxide DMSO solution with a certain concentration into the reaction solution in the step (5); rapidly injecting an ammonium chloride solution with a certain concentration to terminate the coupling reaction; and after continuously stirring for 1-5 hours, stopping stirring, and performing dialysis purification treatment to obtain the final transition metal oxide nano probe.
9. The method for preparing a transition metal oxide nanoprobe according to claim 8, wherein: in the step (1), the feeding mol ratio of biotin to EDC to NHS is 1: e: 1, e is 1-1.2, the mixing reaction time of biotin, EDC and NHS is 0.5-12 hours, and the centrifugal purification condition is that the mixture is centrifuged for 5-30 minutes at 8000-14000 rpm;
in the step (3), the ratio of the biological protein molecules to the activated biological protein molecules is 1: b, b is 5-100, the reaction condition is light-proof and violent stirring, the reaction time is 2-24 hours, the dialysis condition is that a dialysis bag with the cut-off molecular weight of 3000-15000 is selected, and the dialysis time is 1-3 days; in the step (4), the transition metal element solution is a solution containing transition metal ions with a certain concentration, which is obtained by dissolving inorganic salt or acetate of iron, cobalt and nickel, and nitrate in water, wherein the concentration of the transition metal ions is 0.05-1 mol/L, and the ratio of biotin protein molecules to the transition metal elements is 1: c, c is 10-200, stirring and reacting for 1-10 hours after adding the sodium hydroxide aqueous solution, standing and aging for 8-24 hours, and dialyzing the purified transition metal oxide solution under the condition that a dialysis bag with the cut-off molecular weight of 3000-15000 is selected for 1-3 days;
in the step (6), the concentration of DMF or DMSO solution of RBITC or FITC is 2-20 g/L, the molar ratio of RBITC or FITC to protein molecules in the transition metal oxide reaction solution obtained in the step (5) is 1: f, f is 0.1-1, the reaction is completed in a low-temperature environment at 4 ℃, the concentration of ammonium chloride solution is 1-10 mol/L, a dialysis bag with the molecular weight cutoff of 3000-15000 is selected during purification, and the dialysis time is 1-3 days.
10. The transition metal oxide nanoprobe of claim 9The method is applied to a fluorescence imaging experiment and comprises the following steps: when the cell specific protein is subjected to imaging analysis, the selected cell line is digested, planted on a cover glass and fixed, and then the cells are respectively subjected to sealing treatment by using serum albumin and a biotin sealing kit; then carrying out primary antibody incubation for 12 hours with a commercial biotinylated antibody of a specific protein; after incubation, the treated cells were washed three times with PBS and incubated with ChromeoTM546 streptavidin SC-364714 at 37 degrees C environment continued to incubate for 1 hours; and (4) washing with PBS three times, continuously incubating the cells with the nano probe solution obtained in the step (6) for a period of time in an environment at 37 ℃, and performing a fluorescence imaging experiment after washing with PBS three times.
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