CN113639890B - Intracellular assembly method of diamond nanocrystalline and application thereof - Google Patents
Intracellular assembly method of diamond nanocrystalline and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
- G01K11/20—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using thermoluminescent materials
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Abstract
The invention belongs to the technical field of diamond nanocrystals, and discloses an intracellular assembly method of diamond nanocrystals and application thereof. The method comprises the following steps: (1) Mixing the diamond nanocrystalline with a cell culture solution to obtain a diamond nanocrystalline solution; (2) Incubating the diamond nanocrystalline solution and cells together, and removing extra diamond nanocrystalline outside the cells after the incubation is completed to obtain cells containing the diamond nanocrystalline; (3) Applying a light potential well to the cells containing the diamond nanocrystalline, and assembling to obtain diamond nanocrystalline microspheres; the optical power of the optical potential well is 30-200mW, the action time of the optical potential well is 60-200s, and the diameter of the diamond nanocrystalline microsphere is 0.3-2 mu m. The diamond nanocrystalline microsphere assembled by the method has strong stability and excellent fluorescence intensity, and can be used for measuring the temperature of different positions in cells.
Description
Technical Field
The invention belongs to the technical field of diamond nanocrystals, and particularly relates to an intracellular assembly method of a diamond nanocrystal and application thereof.
Background
The organelles within living cells absorb and release heat at specific locations, so detecting the temperature within living cells is important for studying behavior and interactions within cells. In order to realize the temperature measurement in the cells, temperature sensitive materials such as quantum dots, organic dyes, nanogels and the like can be used for measurement, wherein the quantum dots and the organic dyes are easy to swallow in the cells, but have instability due to fluorescence generated by fluorescence scintillation, and have great dependence on local environments (such as pH value, pressure, ion concentration and the like), so the temperature measurement effect is poor; while nanogels can measure the temperature of whole cells, it is difficult to measure the temperature at specific locations of the cells.
The diamond nanocrystalline embedded with the nitrogen vacancy (NV-) has high biocompatibility, stable fluorescence characteristic and sensitive temperature sensing characteristic, and the nano diamond has small size and is easy to enter the inside of a cell, but the signal detection of the single diamond nanocrystalline particles is difficult due to the weak fluorescence intensity, so that the detection capability of the diamond nanocrystalline is improved, and the diamond nanocrystalline particles need to be assembled into the form of diamond nanocrystalline particles. The existing assembly methods of the diamond nanocrystals are various, but each method has defects and shortcomings. For example, self-assembly, while a low cost and simple method, is limited by thermodynamic difficulty in forming stable colloidal structures; the acoustic or magnetic auxiliary method is suitable for large-area assembly and is difficult to implement in single cells; the gold film-based photo-thermal assembly method can achieve manipulation and assembly of single particles, but relying on thermal conversion of gold films limits application in single cells.
Therefore, it is desirable to provide a more efficient, reliable, and stable method of assembling diamond nanocrystals.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides an intracellular assembly method of diamond nanocrystals and application thereof. The method can fully exert the characteristic that the diamond nanocrystalline is easy to enter the inside of the cell, and realize the assembly process of the diamond nanocrystalline in the cell; the assembled diamond nanocrystalline microsphere can not be disassembled, has excellent fluorescence intensity, and can be used for measuring the temperature of different positions in a cell.
The invention provides an intracellular assembly method of diamond nanocrystalline, which comprises the following steps:
(1) Mixing the diamond nanocrystalline with a cell culture solution to obtain a diamond nanocrystalline solution;
(2) Incubating the diamond nanocrystalline solution with cells after sterilization and disinfection, and removing extra diamond nanocrystalline outside the cells after incubation is completed to obtain cells containing the diamond nanocrystalline;
(3) Applying a light potential well to the cells containing the diamond nanocrystalline, and assembling to obtain diamond nanocrystalline microspheres;
the optical power of the optical potential well is 30-200mW, the action time of the optical potential well is 60-200s, and the diameter of the diamond nanocrystalline microsphere is 0.3-2 mu m.
The invention mixes and incubates the diamond nanocrystalline and the cell, so that the diamond nanocrystalline enters the cell through endocytosis. Capturing and moving the diamond nanocrystalline by applying one or more optical potential wells, and assembling the diamond nanocrystalline at different positions (such as positions of different organelles) in the cell to form diamond nanocrystalline microspheres; by arranging the optical potential well array, not only can the diamond nanocrystalline microsphere be assembled, but also a patterned array can be formed. The invention adopts the optical potential well to realize the arrangement and assembly of the diamond nanocrystals in the cell, can assemble the dispersed diamond nanocrystals at any position in the cell to form the diamond nanocrystal microsphere, can strengthen the fluorescence intensity of the diamond nanocrystals, improves the temperature sensing performance of the diamond nanocrystals, and realizes the temperature measurement at different positions in the cell.
Because continuous application of the optical potential well during temperature detection can affect the measurement accuracy, the assembled diamond nanocrystalline microsphere needs to be ensured to be stable and not to be disassembled when the optical potential well is removed. Experiments prove that the diamond nanocrystalline microsphere with large and stable particles can be assembled under the light power and the action time.
Preferably, step (1) further comprises performing ultrasonic vibration on the fluorescent nanodiamond solution. In order to fully disperse the fluorescent nano-diamond in the solution, ultrasonic vibration can be used for promoting the dispersion of the fluorescent nano-diamond.
More preferably, the frequency of the ultrasonic vibration is 20-80KHz.
Preferably, the step (1) further comprises sterilizing the diamond nanocrystalline solution. Since the diamond nanocrystals are assembled in the cell body, the adverse effects of potential microorganisms can be avoided by sterilization and disinfection.
More preferably, the sterilization and disinfection is ultraviolet light treatment.
Preferably, the incubation in step (2) is carried out for a period of 3-6 hours.
The invention also provides application of the intracellular assembly method of the diamond nanocrystalline in measuring cell temperature.
The invention also provides a cell temperature measuring method, which comprises the steps of adopting the intracellular assembly method of the diamond nanocrystalline, and measuring the diamond nanocrystalline microsphere obtained by intracellular assembly through an ultraviolet-visible light-near infrared spectrophotometer to measure the cell temperature.
Compared with the prior art, the invention has the following beneficial effects:
compared with other temperature sensitive materials, the diamond nanocrystalline has high biological compatibility and fluorescence stability. The invention provides the method for assembling and patterning the diamond nanocrystalline microspheres at different positions in a single cell by adopting the optical potential well, and can control the diameter of the formed microsphere, thereby effectively improving the fluorescence intensity and the temperature detection precision of the diamond nanocrystalline and being used for measuring the temperature conditions of different positions in the cell. Meanwhile, the diamond nanocrystalline microsphere assembled by the invention has high stability, and can not be disassembled when the optical potential well is removed.
Drawings
FIG. 1 is a diagram of an experimental apparatus for intracellular assembly and patterned alignment of diamond nanocrystals; the device comprises a laser 1, an acousto-optic deflector 2, a beam expander 3, a dichroic mirror 4, a light filter 5, a water immersion objective 6, a stage 7, a mobile platform 8, an illumination light source 9, an RGB-LED light source 10, a lens 11, a light filter 12, a dichroic mirror 13 and a high-speed CCD camera 14;
FIG. 2 is a graph of experimental results of assembling and patterning diamond nanocrystalline microspheres using a potential well in accordance with the present invention; wherein a is a confocal microscopic imaging diagram of diamond nanocrystalline microspheres with different diameters; b is a fluorescence picture of diamond nanocrystalline microspheres with different diameters; c is a scanning electron micrograph of diamond nanocrystalline microspheres with different diameters; d is an optical imaging picture of arranging diamond nanocrystalline microspheres into a 'NANO' letter pattern in an aqueous solution; e is a dark field fluorescent picture of diamond nanocrystalline microspheres arranged in a 'NANO' pattern and fixed on a glass slide under the excitation of 546nm laser;
FIG. 3 is a statistical plot of experimental data for the assembly of diamond nanocrystalline microspheres using a potential well; wherein a represents an experimental result graph of the diameter of the assembled diamond nanocrystalline microsphere along with the change of laser power under a certain time; b represents an experimental result graph of the diameter of the assembled diamond nanocrystalline microsphere with the change of the assembly time under the fixed capturing power; c represents fluorescence spectrograms of diamond nanocrystalline microspheres with different diameters;
FIG. 4 is a plot of steady state versus capture laser power and capture time for diamond nanocrystalline microspheres; wherein a represents diamond nanocrystalline microspheres formed when the laser is turned on for 20 s; b-d represents the stability condition of the diamond nanocrystalline microsphere after turning on the laser for a certain time and turning off the laser; e represents the time required to maintain the microsphere steady state at different laser powers;
FIG. 5 is a graph of experimental results of assembling and patterning diamond nanocrystalline microspheres within different single cells; wherein a represents confocal microscopy images of different tumor cells; b represents a schematic diagram of the use of an array of optical potential wells spatially arranged in different patterns; c represents a fluorescence diagram of assembling and patterning the diamond nanocrystalline microspheres in different single cells by using a potential well array;
FIG. 6 is an experimental diagram of intracellular temperature detection at different locations after intracellular assembly of diamond nanocrystalline microspheres using an optical potential well; wherein a represents a confocal microscopy image of human brain microvascular endothelial cells (HMEC-1); b represents a dark-field fluorescence plot of mitochondrial staining of human brain microvascular endothelial cells (HMEC-1); c represents fluorescence images of diamond nanocrystals assembled and patterned at different locations in HMEC-1; d represents a fluorescence spectrum diagram of the nano-diamond in solutions with different temperatures, and the inset is an enlarged diagram of Zero Phonon Lines (ZPLs) of the fluorescence spectrum at 20-60 ℃ which are red shifted with the rise of the temperature; e represents an experimental plot of calibration ZPL at each temperature of 20-60 ℃; f represents a fluorescence spectrum of diamond nanocrystalline microspheres near the nuclear membrane and cell membrane of HMEC-1.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments, and any modifications, substitutions, and combinations made without departing from the spirit and principles of the present invention are included in the scope of the present invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1
This example is an experiment of assembling and patterning the diamond nanocrystals. Fig. 1 shows a device used for assembling experiments on diamond nano-crystal particles, as shown in fig. 1, a laser beam emitted by a laser 1 with the wavelength of 1064nm (the laser wavelength is 1064 nm) passes through an acousto-optic deflector 2 (the position and the intensity of an optical potential well can be regulated and controlled; the well position resolution is 0.01 nm), is expanded by a beam expander 3, is upwards reflected by a dichroic mirror 4, enters a water immersion objective lens 6 (the amplification factor is 60 times; the numerical aperture is 1.0) through a light filter 5, and is back-focused to a stage 7 on a moving platform 8 (the moving precision is 60+/-5 nm, and three-dimensional movement can be realized) to form the optical potential well. The light beam 10 (center wavelength: 546 nm) of the RGB-LED light source is coupled into the light path of the microscope through a dichroic mirror 13 after passing through a filter 12, and then is focused on the objective table 7 as excitation light through a water immersion objective 6, the background light used for observing the sample is provided by an illumination light source 9, and the experimental process is photographed by a high-speed CCD camera 14 and monitored on a computer screen in real time.
And (3) at room temperature, preparing a diamond nanocrystalline solution by using diamond nanocrystals with the average diameter of 100+/-30 nm and the number of color centers in single diamond nanocrystals of not less than 300, placing the diamond nanocrystals in an ultrasonic oscillator, and oscillating for 2min at the frequency of 60KHz to enable the diamond nanocrystals to be in a dispersed state in deionized water. Taking the above 0.5mL diamond nanocrystalline solution (diamond nanocrystalline concentration is 2.7X10) 5 /mL) is dropped onto a slide and then placed on a mobile platform 8, using the above-described apparatusAn optical potential well is arranged on the sample, so that the diamond nano-crystal particles are captured to the center of the potential well under the action of optical power. As shown in fig. 2 a (a 1-a 3), under the condition of 60mW of power, with the continuous action of 1064nm of the capturing laser, more diamond nano-crystal particles are gradually captured and assembled to form a stable microsphere structure, and when the action time of the optical potential well is 5s, 7s and 30s, diamond nano-crystal microspheres with diameters of 0.8 μm, 1.5 μm and 2 μm are respectively assembled. The results show that the size of the diamond nanocrystalline microspheres can be adjusted by changing the power and time of capturing laser.
In fig. 2, b (b 1-b 3) and c (c 1-c 3) are respectively a fluorescence image and a scanning electron micrograph image of the diamond nanocrystalline microspheres obtained by the above-mentioned assembly. As the diameter of the assembled microsphere increases, the fluorescence intensity of the assembled microsphere increases, which proves that the assembling method can enhance the fluorescence intensity of the diamond nanocrystalline.
As shown by d in fig. 2, by arranging the optical potential wells controlled by the acousto-optic deflector as an array of optical potential wells, it is possible to realize the assembly of a plurality of nanodiamond microspheres and form a patterned arrangement. And arranging an optical potential well array in the solution, capturing and collecting NANO-diamond to form a letter 'NANO' pattern, gradually approaching to the slide glass direction and approaching to the slide glass by moving the position of the optical potential well array in the z direction, and fixing the assembled microsphere on the slide glass due to Van der Waals force between the microsphere and the slide glass. As shown in fig. 2 e, under 546nm laser irradiation, a "NANO" letter fluorescent image was observed under dark field.
Fig. 3 is a statistical graph of experimental data showing the assembly of diamond nanocrystals using an optical potential well. In fig. 3, a shows a graph of experimental results of the diameter of assembled diamond nanocrystalline microspheres varying with laser power under a certain period of time, and the results show that the diameter of the assembled microspheres becomes larger with the increase of the capturing optical power; fig. 3 b shows a graph of experimental results of the diameter change of assembled diamond nanocrystalline microspheres with the assembly time under the condition of fixed capture power, and the result shows that the diameter of assembled diamond nanocrystalline assembled microspheres increases with the increase of time; in fig. 3, c shows fluorescence spectra of diamond nanocrystalline microspheres with different diameters, and the result shows that the diameter of the diamond nanocrystalline microsphere increases along the advancing direction of the arrow, and the fluorescence intensity also increases.
To verify the relationship between the steady state of the diamond nanocrystalline microspheres and the capturing laser power and capturing time, a laser with a wavelength of 1064nm and a power of 50mW was used, and the diamond nanocrystalline microspheres with a diameter of 2 μm were formed when the laser was applied for 20 seconds (shown in a of FIG. 4). Then the optical potential well is closed after different laser action time is tested, and the dispersion condition of the microsphere is observed. As shown in fig. 4 b and c, when the optical potential well is closed at the action time of 30s and 40s respectively, the microspheres are dispersed, and the assembled microspheres are in an unstable state; as shown in fig. 4 d, when the laser action time reaches 50s, the microsphere maintains a stable spherical structure even if the optical potential well is removed. While the time required to maintain the microsphere steady state at different laser powers is shown in fig. 4 e, which shows that the greater the laser power, the shorter the time required to maintain the microsphere steady state.
In the cell, after the dispersed diamond nano crystal is captured by using 1064nm laser with the power of 60mW to form the microsphere in a stable state, the laser action time is kept to be 100s, then the laser is removed, and the assembled microsphere still maintains the stable state.
Example 2
This example is an experiment of assembling and patterning diamond nanocrystals within different single cells. As shown in FIG. 5 a, the cells used in this example are more typical tumor cells including 4T1 cells (mouse mammary tumor), C127 cells (mouse mammary tumor) and Hela cells (cervical cancer cells) which were cultured in RPMI 1640, DMEM and MEM medium, respectively. The medium was supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin solution (P/S). The cells were then placed in a humidified incubator (temperature: 37 ℃ C.; carbon dioxide concentration: 5%) and grown to a concentration of about 70% over 24 hours, and the cells were washed three times with PBS, and the medium was removed.
The diamond nanocrystals (100.+ -.30 nm) were then mixed with fresh medium to give a solution with a concentration of about 30. Mu.g/mL. After ultraviolet sterilization for 30 minutes, the solution was added to a cell culture dish, and then placed in an incubator (temperature: 37 ℃ C.; carbon dioxide concentration: 5%) for 4 hours, at which time the diamond nanocrystals entered into single cells due to endocytosis. And finally, flushing the cells with a complete culture medium to remove the extracellular diamond nanocrystalline particles. Then stained with Hoechst 33342 dye (Biyun institute of biotechnology, shanghai, excitation light wavelength: 350nm, emission light wavelength: 461 nm) for 30min (final concentration: 2 mg/ml), which binds DNA in the nucleus, the cell activity was characterized by fluorescence intensity of the dye, and after labeling, excess staining reagent was removed by washing with PBS.
The device shown in fig. 1 was used to set up a linear (b 1), rectangular (b 2) and circular (b 3) scanning optical potential well array (1064 nm) as shown in fig. 5 b on three different cell samples, respectively, the capturing optical power of the set optical potential well array was 60mW, the optical potential well array captured diamond nanocrystals dispersed in cytoplasm were assembled into microspheres, and patterned assembled nanodiamond microsphere arrays such as a linear far from the nucleus, a rectangular near the nucleus and a circular array around the nucleus were formed. Irradiation with excitation light (546 nm) gave a fluorescence image of assembled nanodiamond microspheres as shown in fig. 5 c, assembled into microspheres in cells and formed into corresponding patterns (c 1-c 3). In the experiment, the cell nucleus dyed by Hoechst 33342 dye can be excited by irradiation with 350nm laser, normal living cells are round and DNA should be distributed uniformly. As can be seen from FIG. 5 c, the dye distribution in the nuclei is uniform, and the nuclei are rounded indicating that the cell activity is not affected.
Example 3
The present example achieves intracellular temperature detection by measuring fluorescence spectra of diamond nanocrystalline microspheres assembled at different locations within human brain microvascular endothelial cells (HMEC-1). HMEC-1 cells were seeded in confocal dishes of 20mm diameter for 24h, grown to a concentration of about 70%, then washed three times with PBS, and the medium was removed. The diamond nanocrystals (100.+ -.30 nm) were then mixed with fresh medium to a solution at a concentration of about 30. Mu.g/mL. After sterilization with ultraviolet light for 30min, the solution was added to a cell culture dish and then placed in an incubator (temperature: 37 ℃ C.; carbon dioxide concentration: 5%) for 4 hours. Finally, the cells were rinsed with complete medium to remove extracellular diamond nanocrystals, and the observation results of the cells under confocal microscopy are shown in fig. 6 a, with good cell status.
Mitochondria in HMEC-1 cells were then fluorescent stained with Mito-Tracker Green (Biyun institute of biotechnology, shanghai, excitation light wavelength: 490nm, emission light wavelength: 516 nm) for 30min to determine the distribution of mitochondria within the cell, and then washed with medium to remove free staining reagents. After the end of the incubation, the samples were observed with a fluorescence microscope at excitation and emission wavelengths of 490nm and 516nm, respectively, and as a result, mitochondria were mostly concentrated around the nucleus as shown in fig. 6 b.
A plurality of optical potential wells were placed at different locations within the cell using the apparatus shown in fig. 1 to capture and collect diamond nanocrystals dispersed in the cytoplasm and assemble to form diamond nanocrystal microspheres. The diamond nanocrystalline microspheres were arranged at the positions of the nuclear membrane i and the cell membrane ii as shown in fig. 6 c.
Before cell temperature measurement, a temperature calibration curve of the diamond nanocrystals was first obtained. And placing the diamond nanocrystalline in an aqueous solution, controlling the water temperature by using a temperature control table with the precision of 0.5 ℃, performing multiple temperature calibration on the diamond nanocrystalline microspheres in solutions with different temperatures by using an ultraviolet-visible light-near infrared spectrophotometer when the water temperature is stable, and recording the fluorescence spectrum of the diamond nanocrystalline corresponding to the water temperature at 20-60 ℃ and the fluorescence peak position relation changing along with the water temperature. As shown in FIG. 6 d, the NV color center produces a broad emission band with a peak at 685nm and a temperature in the range of 20-60℃with little change in fluorescence intensity, while with further increase in solution temperature, the ZPL (zero phonon line) of electronic transition at 637nm exhibits a red shift. As shown in fig. 6 e, calibration was performed by measuring ZPL at every 5 ℃ of 20 ℃ to 60 ℃, and linear fitting showed a thermal displacement of 25 ℃/nm, thereby obtaining a temperature calibration curve of diamond nanocrystals.
FIG. 6 f shows the use of UV-capable light-near infrared micro-spectrophotometryMeter (CRAIC, 20/30 PV) TM ) Fluorescence spectrum results of diamond nanocrystalline microspheres near the nuclear membrane and cell membrane were measured. The results showed that ZPL moved to 637.8nm and 637.9nm near the nuclear membrane and near the cell membrane, respectively, and that the temperature difference at the positions of nuclear membrane I and cell membrane II was 2.5 ℃ as compared to the temperature calibration curve, indicating that the temperature at the nuclear membrane was higher than the cell membrane, because mitochondria were generally accumulated around the nucleus due to the productivity in normal cells, and the temperature at the nucleus should be higher than other positions, which is consistent with the above-mentioned actual measurement results. The results show that the diamond nanocrystalline microsphere assembled by the invention can carry out intracellular temperature detection at the required position.
The embodiments of the present application have been described in detail above with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the present application and features of the embodiments may be combined with each other without conflict.
Claims (7)
1. An intracellular assembly method of diamond nanocrystals, comprising the steps of:
(1) Mixing the diamond nanocrystalline with a cell culture solution to obtain a diamond nanocrystalline solution;
(2) Incubating the diamond nanocrystalline solution and cells together, and removing extra diamond nanocrystalline outside the cells after the incubation is completed to obtain cells containing the diamond nanocrystalline;
(3) Applying a light potential well to the cells containing the diamond nanocrystalline, and assembling to obtain diamond nanocrystalline microspheres;
the optical power of the optical potential well is 30-200mW, the action time of the optical potential well is 60-200s, and the diameter of the diamond nanocrystalline microsphere is 0.3-2 mu m.
2. The method of intracellular assembly of diamond nanocrystals according to claim 1, wherein step (1) further comprises subjecting the diamond nanocrystal solution to ultrasonic vibration.
3. The method of intracellular assembly of diamond nanocrystals according to claim 2, wherein the frequency of the ultrasonic oscillation is 20-80KHz.
4. The method of intracellular assembly of diamond nanocrystals according to claim 1, wherein step (1) further comprises sterilizing the diamond nanocrystal solution.
5. The method of intracellular assembly of diamond nanocrystals according to claim 1, wherein the incubation time of step (2) is 3-6 hours.
6. Use of the method of intracellular assembly of diamond nanocrystals according to any one of claims 1 to 5 for determining cell temperature.
7. A method for measuring cell temperature, wherein the method for assembling diamond nanocrystals according to any one of claims 1 to 5 is followed by measuring the diamond nanocrystal microsphere obtained by intracellular assembly by an ultraviolet-visible-near infrared spectrophotometer to measure cell temperature.
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