CN113639890A - Intracellular assembly method of diamond nanocrystals and application thereof - Google Patents
Intracellular assembly method of diamond nanocrystals and application thereof Download PDFInfo
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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 nanocrystal solution and cells together, and removing redundant diamond nanocrystals outside the cells after incubation is finished to obtain cells containing the diamond nanocrystals; (3) applying a light potential well to the cells containing the diamond nanocrystals, and assembling to obtain diamond nanocrystal 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 μm. The diamond nanocrystalline microspheres assembled by the method have high 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 diamond nanocrystals and application thereof.
Background
Organelles in living cells absorb and release heat at specific locations, and therefore, detecting the temperature within a living cell is very important for studying behavior and interactions within the cell. In order to realize intracellular temperature measurement, 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 into cells, but have poor temperature measurement effect because fluorescence generated by fluorescence scintillation is unstable and has great dependence on local environments (such as pH value, pressure, ion concentration and the like); while nanogels can measure the temperature of the entire cell, it is difficult to measure the temperature of a specific location of the cell.
The diamond nanocrystalline embedded with the nitrogen vacancy (NV-) has high biocompatibility, stable fluorescence characteristic and sensitive temperature sensing characteristic, the size of the nanodiamond is small, the nanodiamond easily enters cells, but the signal detection is difficult due to the fact that the fluorescence intensity of single diamond nanocrystalline particles is weak, and the diamond nanocrystalline particles need to be assembled into the form of diamond nanocrystalline particles in order to improve the detection capability of the diamond nanocrystalline. There are many methods for assembling diamond nanocrystals, but each method has its drawbacks and deficiencies. For example, self-assembly, while a low cost and simple process, is limited by the thermodynamic difficulties in forming stable colloidal structures; the acoustic or magnetic auxiliary method is suitable for large-area assembly and is difficult to implement in a single cell; the gold film-based photothermal assembly method can realize the manipulation and assembly of single particles, but the thermal conversion depending on the gold film limits the application in single cells.
Therefore, it is desirable to provide a more efficient, reliable and stable method for assembling diamond nanocrystals.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides an intracellular assembly method of diamond nanocrystals and application thereof. By adopting the method, the characteristic that the diamond nanocrystalline is easy to enter the interior of the cell can be fully exerted, and the assembling process of the diamond nanocrystalline is realized in the interior of the cell; the assembled diamond nanocrystalline microspheres cannot be disintegrated, have excellent fluorescence intensity and can be used for measuring the temperature of different positions in cells.
The invention provides an intracellular assembly method of diamond nanocrystals, which comprises the following steps:
(1) mixing the diamond nanocrystalline with a cell culture solution to obtain a diamond nanocrystalline solution;
(2) sterilizing and disinfecting the diamond nanocrystal solution, incubating the sterilized and disinfected diamond nanocrystal solution with cells, and removing redundant diamond nanocrystals outside the cells after incubation is finished to obtain the cells containing the diamond nanocrystals;
(3) applying a light potential well to the cells containing the diamond nanocrystals, and assembling to obtain diamond nanocrystal 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 μm.
According to the invention, the diamond nanocrystals and cells are mixed and incubated, so that the diamond nanocrystals enter the cells through endocytosis. Then, by applying one to a plurality of light potential wells, the diamond nanocrystalline is captured and moved, and the diamond nanocrystalline microspheres are assembled at different positions (such as positions of different organelles) in cells; by arranging the light potential trap array, the diamond nanocrystalline microspheres can be assembled, and a patterned array can be formed. The invention adopts the optical potential well to realize the arrangement and the assembly of the diamond nanocrystalline in the cell, can assemble the dispersed diamond nanocrystalline at any position in the cell to form the diamond nanocrystalline microsphere, can strengthen the fluorescence intensity of the diamond nanocrystalline, improves the temperature sensing performance of the diamond nanocrystalline, and realizes the temperature measurement at different positions in the cell.
Because the continuous application of the light potential well during the temperature detection can affect the determination accuracy, the assembled diamond nanocrystalline microspheres can be kept stable without disintegration when the light potential well is removed. According to the invention, the diamond nanocrystalline microspheres with large and stable particles can be assembled under the optical power and action time.
Preferably, the step (1) further comprises subjecting the fluorescent nanodiamond solution to ultrasonic oscillation. In order to fully disperse the fluorescent nano-diamond in the solution, ultrasonic oscillation can be adopted to promote the dispersion.
More preferably, the frequency of the ultrasonic oscillation is 20-80 KHz.
Preferably, the step (1) further comprises sterilizing the diamond nanocrystalline solution. Since the diamond nanocrystals need to be assembled in cells, sterilization can be performed to avoid the adverse effects of potential microorganisms.
More preferably, the sterilization is an ultraviolet light treatment.
Preferably, the incubation time in step (2) is 3-6 h.
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 is characterized in that after the diamond nanocrystalline intracellular assembly method is adopted, the diamond nanocrystalline microspheres obtained by intracellular assembly are measured by 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 nanocrystals have high biocompatibility and fluorescence stability. The invention provides a method for assembling and patterning diamond nanocrystalline microspheres at different positions in a single cell by using a light potential well, and the method can control the diameter of the formed microspheres, effectively improve the fluorescence intensity and the temperature detection precision of the diamond nanocrystalline, and can be used for measuring the temperature conditions of different positions in the cell. Meanwhile, the diamond nanocrystalline microspheres assembled by the method have high stability, and can not be disintegrated when the light potential well is removed.
Drawings
FIG. 1 is a diagram of an experimental apparatus for assembling and patterning diamond nanocrystals in a cell; 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 lens 6, an objective table 7, a moving 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 the experimental results of assembling and patterning diamond nanocrystalline microspheres using a photo-potential trap in accordance with the present invention; wherein a is a confocal microscopic imaging picture of diamond nanocrystalline microspheres with different diameters; b is a fluorescence picture of the 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 in which the diamond nanocrystalline microspheres are arranged into 'NANO' letter patterns in an aqueous solution; e, arranging the diamond nanocrystalline microspheres into 'NANO' patterns, fixing the NANO patterns on a glass slide, and carrying out dark field fluorescence picture under the excitation of 546nm laser;
FIG. 3 is a statistical chart of experimental data for assembling diamond nanocrystalline microspheres using a photo potential trap; wherein a represents an experimental result curve graph of the diameter of the assembled diamond nanocrystalline microsphere changing along with the laser power under a certain time; b represents an experimental result curve graph of the diameter of the assembled diamond nanocrystalline microspheres changing along with the assembly time under the fixed capture power; c represents a fluorescence spectrum of the diamond nanocrystalline microspheres with different diameters;
FIG. 4 is a relationship between the steady state of diamond nanocrystalline microspheres and the trapping laser power and trapping time; wherein a represents diamond nanocrystalline microspheres formed when laser action is turned on for 20 s; b-d represents the stability condition of the diamond nanocrystalline microspheres after the laser action is started for a certain time and then is closed; e represents the time required to maintain the microsphere in a stable state under different laser powers;
FIG. 5 is a graph of experimental results of assembling and patterning diamond nanocrystalline microspheres in different single cells; wherein a represents confocal microscopy images of different tumor cells; b represents a schematic diagram of different patterns spatially arranged by using a light potential well array; c represents a fluorescence image of assembling and patterning arrangement of the diamond nanocrystalline microspheres in different single cells by using a light potential well array;
FIG. 6 is an experimental diagram of intracellular temperature detection at different positions after assembling diamond nanocrystalline microspheres in cells by using optical potential wells; wherein a represents a confocal micrograph of human brain microvascular endothelial cells (HMEC-1); b represents a dark-field fluorescence map of mitochondrial staining of human brain microvascular endothelial cells (HMEC-1); c represents a fluorescence image of the diamond nanocrystals assembled and patterned at different positions in the HMEC-1; d represents the fluorescence spectrogram of the nano-diamond in the solution at different temperatures, and the inset is an enlarged view of the red shift of Zero Phonon Lines (ZPLs) of the fluorescence spectrum along with the temperature rise at 20-60 ℃; e represents experimental graphs for calibrating ZPL at each temperature of 20-60 ℃; f represents a fluorescence spectrum for measuring the diamond nanocrystalline microspheres near the nuclear membrane and the cell membrane of the 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 are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1
This example is an experiment in which diamond nanocrystals were assembled and patterned. Fig. 1 shows a device used for an assembly experiment of diamond nanocrystalline particles, as shown in fig. 1, a laser beam emitted by a laser 1 (laser wavelength: 1064nm) with a wavelength of 1064nm passes through an acousto-optic deflector 2 (the position and intensity of a light potential well can be adjusted and controlled; the well site resolution: 0.01nm), is expanded by a beam expander 3, is reflected upwards by a dichroic mirror 4, enters a water immersion objective lens 6 (the magnification is 60 times; the numerical aperture: 1.0) through a filter 5, and is focused on an objective table 7 of a moving platform 8 (the moving precision: 60 ± 5nm, which can realize three-dimensional movement) to form the light potential well. Light beams 10 (central wavelength: 546nm) of an RGB-LED light source are coupled into a light path of a microscope through a dichroic mirror 13 after passing through a light filter 12, then are focused on an objective table 7 through a water immersion inverted objective 6 to be used as exciting light, background light for observing a sample is provided by an illuminating light source 9, an experimental process is shot by a high-speed CCD camera 14, and real-time monitoring is carried out on a computer screen.
And at room temperature, preparing the diamond nanocrystals with the average diameter of 100 +/-30 nm and the number of color centers of not less than 300 in a single diamond nanocrystal into a diamond nanocrystal solution, placing the diamond nanocrystal solution in an ultrasonic oscillator, and oscillating the diamond nanocrystal solution for 2min at the frequency of 60KHz to ensure that the diamond nanocrystals are in a dispersed state in deionized water. The above 0.5mL of diamond nanocrystal solution (diamond nanocrystal concentration of 2.7X 10)5mL) on a slide, and then placed on a moving platform 8, and a light potential well is arranged on the sample by using the device, so that the diamond nanocrystalline particles are captured to the center of the potential well under the action of light force. As shown in a (a1-a3) in FIG. 2, under the condition of a power of 60mW, more diamond nanocrystalline particles are gradually captured and assembled to form a stable microsphere structure with the continuous action of a capture laser 1064nm, and diamond nanocrystalline microspheres with the diameters of 0.8 μm, 1.5 μm and 2 μm are assembled when the action time of the potential trap is respectively 5s, 7s and 30 s. The above results show that the size of the diamond nanocrystalline microspheres can be adjusted by changing the power and time of the trapping laser.
In FIG. 2, b (b1-b3) and c (c1-c3) are respectively a fluorescence picture and a scanning electron microscope picture of the assembled diamond nanocrystalline microspheres. The fluorescent intensity of the assembled microspheres is enhanced along with the increase of the diameter of the assembled microspheres, and the fact that the fluorescent intensity of the diamond nanocrystals can be enhanced by the assembling method is proved.
As shown in d of fig. 2, the light potential trap controlled by the acousto-optic deflector is arranged as a light potential well array, so that the assembly of a plurality of nano-diamond microspheres can be realized, and a patterned arrangement can be formed. The method comprises the steps of arranging a photo potential well array in a solution, capturing and collecting NANO-diamonds to form a character 'NANO' pattern, gradually approaching to a slide glass and being close to the slide glass by moving the position of the photo potential well array in the z direction, and fixing assembled microspheres on the slide glass due to van der Waals force action between the microspheres and the slide glass. As shown in fig. 2, e, under 546nm laser illumination, a "NANO" letter fluorescence image can be observed under dark field.
Fig. 3 is a statistical chart of experimental data for assembling diamond nanocrystals using a potential well. Wherein, a in fig. 3 shows the experimental result curve graph of the diameter of the assembled diamond nanocrystalline microspheres changing with the laser power under a certain time, and the result shows that the diameter of the assembled microspheres becomes larger with the increase of the captured light power; b in fig. 3 shows a graph of experimental results of the diameter change of the assembled diamond nanocrystalline microspheres with the assembly time under the fixed capture power, and the results show that the diameter of the assembled diamond nanocrystalline assembled microspheres increases with the increase of the time; the fluorescence spectrum of diamond nanocrystalline microspheres with different diameters is shown in c in fig. 3, and the result shows that the diameter of the diamond nanocrystalline microspheres is increased along the arrow direction, and the fluorescence intensity is increased accordingly.
In order to verify the relationship between the steady state of the diamond nanocrystalline microspheres and the trapping laser power and trapping time, a laser with a wavelength of 1064nm and a power of 50mW was used to form diamond nanocrystalline microspheres with a diameter of 2 μm (shown as a in FIG. 4) when applied for 20 s. And then testing to close the light potential well after different laser action time, and observing the dispersion condition of the microspheres. As shown in b and c in fig. 4, when the action time is 30s and 40s respectively, the light potential trap is closed, the microspheres are dispersed, and the assembled microspheres are in an unstable state; when the laser exposure time reached 50s, the microspheres remained in a stable spherical configuration even with the optical potential well removed, as shown by d in fig. 4. While e in fig. 4 shows the time required to maintain the microsphere in a stable state at different laser powers, the results show that the greater the laser power, the shorter the time required to maintain the microsphere in a stable state.
In the cell, after a 1064nm laser with the power of 60mW is used for capturing and dispersing the diamond nano-crystalline form into microspheres in a stable state, the action time of the laser is kept to reach 100s, then the laser is removed, and the assembled microspheres still maintain the stable state.
Example 2
This example is an experiment of assembling and patterning diamond nanocrystals within different single cells. As shown in a in fig. 5, the cells used in the present example are typical tumor cells including 4T1 cells (mouse mammary tumor), C127 cells (mouse mammary tumor) and Hela cells (cervical cancer cells), and the tumor cells were cultured in RPMI 1640, DMEM and MEM media, respectively. The medium was supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin solution (P/S). The cells were then put into a humidified incubator (temperature: 37 ℃ C.; carbon dioxide concentration: 5%) and increased to a concentration of about 70% in 24 hours, and then the cells were washed three times with PBS to remove the medium.
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%) to be cultured for 4 hours, at which time the diamond nanocrystals entered into single cells due to endocytosis. Finally, the cells are washed by using a complete culture medium, and the diamond nanocrystalline particles outside the cells are removed. Then, the cells were stained with Hoechst 33342 dye (Biyunstian institute of biotechnology, Shanghai, excitation light wavelength: 350nm, emission light wavelength: 461nm) which binds to DNA in the nuclei for 30min (final concentration: 2mg/ml), the cell activity was characterized by the fluorescence intensity of the dye, and after labeling, excess staining reagent was removed by washing with PBS.
Linear (b1), rectangular (b2) and circular (b3) scanning light potential well arrays (1064nm) as shown in b in fig. 5 were respectively arranged on three different cell samples by using the device shown in fig. 1, the capture light power of the potential well arrays was set to be 60mW, the potential well arrays captured diamond nanocrystals dispersed in cytoplasm to assemble microspheres, and patterned assembled nanodiamond microsphere arrays were formed, such as linear far from cell nucleus, rectangular near cell nucleus and circular array around cell nucleus. The fluorescence image of the assembled nanodiamond microspheres shown as c in fig. 5 was obtained by irradiation with excitation light (546nm), assembled into microspheres inside the cells and formed into the corresponding pattern (c1-c 3). In the experiment, the 350nm laser is used for irradiating, the cell nucleus stained by Hoechst 33342 dye can be excited, normal living cells are round, and DNA is distributed uniformly. As can be seen from c in FIG. 5, the dye distribution in the nuclei was uniform, and the rounded shape of the nuclei indicates that the cell activity was not affected.
Example 3
In the embodiment, the intracellular temperature detection is realized by measuring the fluorescence spectrum of the diamond nanocrystalline microspheres assembled at different positions in human brain microvascular endothelial cells (HMEC-1). HMEC-1 cells were seeded in a 20mm diameter confocal culture dish for 24h, grown to a concentration of about 70%, and then washed three times with PBS to remove the medium. The diamond nanocrystals (100. + -.30 nm) were then formulated with fresh medium into a solution having a concentration of about 30. mu.g/mL. After ultraviolet sterilization for 30min, the solution was added to a cell culture dish, and then placed in an incubator (temperature: 37 ℃ C.; carbon dioxide concentration: 5%) to be cultured for 4 hours. Finally, the cells were washed with complete medium to remove the diamond nanocrystals outside the cells, and the observation results of the cells under a confocal microscope are shown in a in fig. 6, indicating that the cells were in good condition.
Then, mitochondrion was subjected to fluorescent staining for 30min in HMEC-1 cells using Mito-Tracker Green (Biyuntian Biotech institute, Shanghai, excitation light wavelength: 490nm, emission light wavelength: 516nm) to determine the intracellular distribution position of the mitochondrion, and then free staining reagent was removed by washing with a medium. After the incubation, the sample was 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 b in FIG. 6.
A plurality of light potential wells are arranged at different positions in the cell by using the device shown in figure 1 so as to capture and collect the dispersed diamond nanocrystals in cytoplasm, and the diamond nanocrystals are assembled to form the diamond nanocrystal microspheres. The diamond nanocrystalline microspheres were arranged at the locations of the nuclear membrane i and the cell membrane ii as shown in c in fig. 6.
Before cell temperature measurement, a temperature calibration curve of diamond nanocrystals was first obtained. 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 temperature calibration on the diamond nanocrystalline microspheres in the solution at different temperatures for multiple times 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 changed along with the water temperature. As shown in d of FIG. 6, the NV color center produces a broad emission band with a peak at 685nm and a temperature in the range of 20-60 deg.C, with little change in fluorescence intensity, and with further increase in solution temperature, ZPL (zero phonon line) at an electronic transition at 637nm exhibits a red shift. As shown in e of fig. 6, calibration was performed by measuring ZPL at every 5 ℃ of 20 ℃ to 60 ℃, and linear fitting revealed that the thermal displacement was 25 ℃/nm, thereby obtaining a temperature calibration curve of diamond nanocrystals.
In FIG. 6 f is shown the use of a UV-visible light-near infrared microspectrophotometer (CRAIC, 20/30 PV)TM) And measuring the fluorescence spectrum result of the diamond nanocrystalline microspheres close to the cell nuclear membrane and the cell membrane. The results show that ZPL moves to 637.8nm and 637.9nm near the nuclear membrane and near the cell membrane, respectively, and compared to the temperature calibration curve, a temperature difference of 2.5 ℃ can be obtained at the nuclear membrane I and cell membrane II locations, indicating that the temperature at the nuclear membrane is higher than that of the cell membrane, because mitochondria are usually accumulated around the nucleus in normal cells, and the temperature at the nucleus should be higher than other locations, which is consistent with the above actual measurement results. The results show that the diamond nanocrystalline microspheres assembled by the method can be used for detecting the intracellular temperature at a required position.
The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
Claims (7)
1. An intracellular assembly method of diamond nanocrystals is characterized by comprising the following steps:
(1) mixing the diamond nanocrystalline with a cell culture solution to obtain a diamond nanocrystalline solution;
(2) incubating the diamond nanocrystal solution and cells together, and removing redundant diamond nanocrystals outside the cells after incubation is finished to obtain cells containing the diamond nanocrystals;
(3) applying a light potential well to the cells containing the diamond nanocrystals, and assembling to obtain diamond nanocrystal 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 μm.
2. The method for intracellular assembly of diamond nanocrystals, as recited in claim 1, wherein the step (1) further comprises subjecting the diamond nanocrystal solution to ultrasonic agitation.
3. The method for intracellular assembly of diamond nanocrystals, according to claim 2, wherein the frequency of the ultrasonic oscillation is 20 to 80 KHz.
4. The method for intracellular assembly of diamond nanocrystals, as recited in claim 1, wherein the step (1) further comprises sterilizing the diamond nanocrystal solution.
5. The method for intracellular assembly of diamond nanocrystals, as recited in claim 1, wherein the incubation time of step (2) is 3-6 h.
6. Use of a method of intracellular assembly of diamond nanocrystals according to any one of claims 1 to 5 in the determination of cell temperature.
7. A method for measuring cell temperature, comprising the step of measuring the cell temperature by measuring diamond nanocrystal microspheres obtained by intracellular assembly using the method for intracellular assembly of diamond nanocrystals according to any one of claims 1 to 5 using an ultraviolet-visible-near infrared spectrophotometer.
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