CN115192606B - Monoatomic nano-enzyme Pt@MoS 2 Preparation method and application thereof - Google Patents
Monoatomic nano-enzyme Pt@MoS 2 Preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a monoatomic nano enzyme Pt@MoS 2 And the preparation method and application thereof, the preparation method comprises the following steps: mixing potassium thiocyanate and ammonium molybdate tetrahydrate, preserving heat for 1.5-2.5 h at the temperature of 250-350 ℃ to obtain black solid, washing the black solid with water, and drying to obtain MoS 2 MoS is to 2 Uniformly mixing the solution with a solvent to obtain a dispersion liquid, dripping the dispersion liquid on a conductive carrier, and drying to obtain a substrate; injecting Pt metal plasma into the surface of the substrate, on which the dispersion liquid is dripped, for 80-150 min to obtain the monoatomic nano enzyme Pt@MoS 2 . The monoatomic nano enzyme Pt@MoS synthesized by the preparation method of the invention 2 Proved by the ion implantation method, the single-atom effective load can be realized and the proliferation of 4T1 cells can be inhibited, on one hand, the active site of noble metal single-atom Pt can be effectively utilized to catalyze the oxidation stress reaction of cancer cells to induce a large number of apoptosis of the cancer cells, and the single-atom nano enzyme Pt@MoS is realized 2 Inhibiting proliferation of breast cancer cells of mice; another aspect provides an ion implantation method that can achieve a single atom Pt payload.
Description
Technical Field
The invention belongs to the technical field of anti-tumor of monoatomic materials, and particularly relates to monoatomic nano enzyme Pt@MoS 2 And a preparation method and application thereof.
Background
Nanoezymes have been widely developed for their unique physicochemical properties and catalytic activities, but their catalytic activities are far lower than those of natural enzymes. The single-atom nano-enzyme has a definite electronic geometry, and the single-atom nano-enzyme has excellent catalytic performance in various catalytic reactions by using isolated metal atom active sites, can simulate the internal catalytic active center of the natural enzyme so as to solve the limitations of poor stability, high cost, difficult storage and the like of the natural enzyme, and becomes a direct substitute of the traditional enzyme.
The monoatomic nanoenzymes are typically prepared using chemical synthesis, atomic force deposition, one-pot processes, and the like. However, the methods for preparing these monoatomic nanoenzymes (SAzymes) generally often lack the proper interactions between the metal active atoms and the carrier, resulting in instability and leaching of the active species. In particular, due to the high specific surface free energy, the individual metal atoms have a strong tendency to migrate and agglomerate into particles, i.e. sinter, resulting in a decrease or even deactivation of the catalytic properties of the SAzymes. Therefore, a new technology is needed to expand the preparation mode of the single-atom material, and the defects of structural instability, low utilization rate of metal active sites and the like of the single-atom material are improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a monoatomic nano-enzyme Pt@MoS 2 The preparation method comprises the steps of injecting metal Pt ions with a certain energy range into molybdenum disulfide by using a high-energy metal ion implanter to synthesize monoatomic nano enzyme Pt@MoS 2 。
Another object of the present invention is to provide the monoatomic nano-enzyme Pt@MoS obtained by the above preparation method 2 。
The aim of the invention is achieved by the following technical scheme.
Monoatomic nano-enzyme Pt@MoS 2 The preparation method of (2) comprises the following steps:
1) Mixing potassium thiocyanate and ammonium molybdate tetrahydrate, preserving heat for 1.5-2.5 h at the temperature of 250-350 ℃ to obtain black solid, washing the black solid with water, and drying to obtain MoS 2 Wherein, the ratio of potassium thiocyanate to ammonium molybdate tetrahydrate is (3-6) according to the mass parts: (0.1 to 0.2);
in the step 1), the drying temperature is 60-80 ℃, and the drying time is 18-24 hours.
In the step 1), after mixing potassium thiocyanate and ammonium molybdate tetrahydrate, the temperature is raised from room temperature of 20 to 25 ℃ to 250 to 350 ℃ at a rate of 5 to 10 ℃/min.
2) MoS is carried out 2 And a solvent are uniformly mixed to obtain a dispersion liquid, wherein the solvent is a mixture of absolute ethyl alcohol and deionized water, the ratio of the absolute ethyl alcohol to the deionized water is 1 (3-4) in parts by volume, and MoS in the dispersion liquid 2 The concentration of (C) is 0.006-0.0075 g/mL;
3) Dropping the dispersed liquid on a conductive carrier, and drying to obtain a substrate;
in the step 3), the conductive carrier is a metal sheet or a conductive glass sheet.
In the step 3), moS is added to the dispersion liquid dropwise per square centimeter of the conductive carrier 2 The mass of (2) is 0.0025-0.005 g.
In the step 3), the drying temperature is 60-80 ℃, and the drying time is 10-12 h.
4) Injecting Pt metal plasma into the surface of the substrate, on which the dispersion liquid is dripped, for 80-150 min to obtain the monoatomic nano enzyme Pt@MoS 2 。
In the step 4), a high-energy ion implanter is used for implanting Pt metal plasma.
In the step 4), the high energy ion implanter is a MEVVA type source.
In the step 4), after the cathode high-voltage pulse of the high-energy ion implanter is triggered, pt metal plasma is led out from an anode Pt target.
In the step 4), the vacuum degree in the chamber for injecting Pt metal plasma by the high-energy ion implanter is lower than 6 multiplied by 10 -3 pa。
In the step 4), the purity of the anode Pt target is more than or equal to 99.99wt%.
In the step 4), the triggering frequency of the cathode high-voltage pulse is 8-15 Hz, the triggering voltage is 3-6 kV, the arc voltage is 40-150V, the arc current is 0.4-1.5A, the inhibiting voltage is 0.6-1.1 kV, the injection voltage is 10-50 kV, and the extraction current is 0.2-0.5A.
In the step 4), after the injection of the Pt metal plasma is completed, the substrate is cleaned by absolute ethyl alcohol, and the cleaning method comprises the following steps: immersing the substrate in absolute ethyl alcohol, performing ultrasonic treatment for 1.5-2 h, performing vacuum filtration, and drying the solid obtained by vacuum filtration at 60-80 ℃ for 10-12 h.
The monoatomic nano-enzyme Pt@MoS obtained by the preparation method 2 。
The monoatomic nano-enzyme Pt@MoS 2 The application of the composition in inhibiting proliferation of mouse breast cancer cells (4T 1).
The monoatomic nano enzyme Pt@MoS synthesized by the preparation method of the invention 2 Proved by the ion implantation method, the single-atom effective load can be realized and the proliferation of 4T1 cells can be inhibited, on one hand, the active site of noble metal single-atom Pt can be effectively utilized to catalyze the oxidation stress reaction of cancer cells to induce a large number of apoptosis of the cancer cells, and the single-atom nano enzyme Pt@MoS is realized 2 Inhibition of 4T1 cell proliferation; another aspect provides an ion implantation method that can achieve a single atom Pt payload.
Drawings
FIG. 1 shows (a) MoS obtained by comparative example 2 High-power TEM image of (b) MoS obtained by comparative example 2 High-order lattice spacing diagram (the inset is the corresponding Fourier transform image), (c) comparative example preparation of the resulting MoS 2 Selected area electron diffraction patterns (SEAD), (d) comparative example preparation of the resulting MoS 2 High-power TEM image of (a) and corresponding HAADF-TEM image, EDS element map of Mo and S element (scale: 100 nm), (e) monoatomic nanoenzyme Pt@MoS prepared in example 1 2 TEM image of (f) the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 HRTEM lattice spacing pattern (inset is corresponding fourier transform image), (g) monoatomic nanoenzyme pt@mos prepared in example 1 2 Selected area electron diffraction patterns (SEAD), (h) the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 HAADF-TEM image of (and corresponding EDS element map of Mo, S, pt elements (scale: 100 nm);
FIG. 2 shows the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 A spherical aberration electron microscope HAADF-STEM diagram;
FIG. 3 shows (a) Pt foil and the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 Pt L of (2) 3 Edge normalized XANES spectrum, (b) Pt foil and monoatomic nano enzyme Pt@MoS prepared in example 1 2 Fourier transform EXAFS spectrum in R space and (c) monoatomic nanoenzyme Pt@MoS prepared in example 1 2 XPS fine spectra of Pt 4 f;
FIG. 4 shows the MoS obtained by the preparation of comparative example (control) 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Spin-trapping ESR spectra in DMPO under the same conditions;
FIG. 5 shows (a) MoS obtained by comparative example 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Variation of 4T1 cell proliferation inhibition ratio at 200 μg/mL with different action time (0 h,2h,6h,12h,24 h), (b) MoS prepared by comparative example 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Changes in proliferation inhibition of 4T1 cells (P) at different concentrations of action (50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) for 24 hours<0.0001，**P<0.005，*P<0.05, n=5 independent experimental groups, values mean ± SEM);
FIG. 6 shows the MoS obtained in the comparative example 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Calcein-AM and PI co-stained laser co-microscope images of 4T1 cells (scale: 100 μm).
Detailed Description
The preparation method of the present invention will be described in detail with reference to the accompanying drawings.
The following related sources of purchase for the drugs are as follows:
potassium thiocyanate: KSCN,99%,3A;
ammonium molybdate tetrahydrate: (NH) 4 ) 6 Mo 7 O 24 ·4H 2 O,99%, aladine.
The following instruments and models are involved:
muffle furnace: LFM1200C, joint fertilizer Kang Pa, equipment technologies limited.
Vacuum oven: DZF-6050, synechocyst materials science and technology Co.
The pure titanium sheet is cleaned before use: hydrochloric acid (Allatin, 12 mol/L), acetone (Allatin), absolute ethanol (Allatin) and deionized water are sequentially used, and an ultrasonic cleaner (KQ 3200E, kunshan ultrasonic instruments Co., ltd.) is used for soaking and cleaning for 3 times for 30min each time; cleaning, and drying in a vacuum oven at 80 ℃.
Cell complete medium configuration: 89wt% RPMI-1640 (BIOIND), 10wt% Australian fetal bovine serum (FBS, gibco) and 1wt% penicillin and streptomycin antibacterial (HyClone) mixture.
Example 1
The method comprises the following steps ofMonoatomic nano-enzyme Pt@MoS 2 The preparation method of (2) comprises the following steps:
1) The potassium thiocyanate was weighed using an electronic weighing balance (XSE 105, metrele-Tolydoku Shanghai Co., ltd.), 5g of potassium thiocyanate and 0.15g of ammonium molybdate tetrahydrate were mixed in a crucible, the crucible was placed in a muffle furnace, the temperature was raised to 300℃from room temperature of 20 to 25℃at a rate of 5℃per minute, and the temperature was maintained at 300℃for 2 hours to obtain a black solid, which was uniformly dispersed with deionized water to obtain a mixed solution, and the mixed solution was purified by a circulating water type vacuum pump (SHZ-DIII, incorporated by Ware instruments Co., ltd.) at 1X 10 -1 Filtering the mixed solution under the vacuum degree Pa, and putting the solid obtained by filtering into a vacuum oven to dry for 24 hours at 80 ℃ to obtain MoS 2 Wherein, uniformly dispersing black solids with deionized water is to rinse off excess KSCN (ignoring the effects of carbon and oxygen elements on the material);
2) Will be 0.03g MoS 2 Mixing with a solvent, and carrying out ultrasonic treatment for 120min until the solvent is uniformly mixed to obtain a dispersion, wherein the solvent is a mixture of absolute ethyl alcohol and deionized water, the ratio of the absolute ethyl alcohol to the deionized water is 1:3 in parts by volume, and MoS is contained in the dispersion 2 Is 0.0075g/mL;
3) Pure titanium sheet (length x width x thickness: 15 mm. Times.15 mm. Times.10 mm) as a conductive carrier, dropping the dispersion on the conductive carrier with a 200. Mu.l pipette, and drying at 80℃for 12 hours to obtain a substrate, wherein MoS is added dropwise to the dispersion per square centimeter of the conductive carrier 2 The mass of the titanium alloy is 0.0025g, and the dispersion liquid can be uniformly distributed on the surface of the pure titanium sheet to form MoS due to the surface tension of the liquid 2 Dense layer, and does not change MoS 2 A chemical structure;
4) Injecting Pt metal plasma into the surface of the substrate with the dispersion liquid by using a high-energy ion implanter (MEWA 80 III-100, china, MEVVA type source) for 90min, immersing pure titanium sheet into absolute ethyl alcohol after injecting Pt metal plasma, performing ultrasonic treatment for 2h to disperse powder on the substrate in the absolute ethyl alcohol, vacuum-filtering the absolute ethyl alcohol with the dispersed powder, and drying the solid obtained by filtering at 80 ℃ for 12h to obtain the monoatomic nano enzyme Pt@MoS 2 Wherein, the cathode high-voltage pulse of the high-energy ion implanter is triggered and led out from the anode Pt targetPt metal plasma, and vacuum degree in chamber for injecting Pt metal plasma by high-energy ion implanter when injecting Pt metal plasma is 4×10 -3 pa, the purity of the anode Pt target was 99.99wt%. The trigger frequency of the cathode high-voltage pulse is 10Hz, the trigger voltage is 4kV, the arc voltage is 50V, the arc current is 0.4-1.5A (automatically regulated by the high-energy ion implanter in the range), the suppression voltage is 1kV, the injection voltage is 40kV, and the extraction current is 0.2-0.5A (automatically regulated by the high-energy ion implanter in the range).
Example 2
Monoatomic nano-enzyme Pt@MoS 2 The preparation method of (2) was substantially the same as that of example 1, except that the injection voltage was 30kV in this example.
Example 3
Monoatomic nano-enzyme Pt@MoS 2 The preparation method of (2) was substantially the same as that of example 1, except that the injection voltage was 20kV in this example.
Comparative example
MoS (MoS) 2 See step 1 in example 1 for a preparation method thereof).
Comparative examples and examples 1 to 3 were subjected to X-ray photoelectron spectroscopy (XPS) to obtain the relative atomic ratios of Mo, S and Pt elements, and specific test results are shown in table 1.
TABLE 1
Microscopic surface morphology of the comparative examples was observed by Transmission Electron Microscopy (TEM) as shown in fig. 1 a-d. As shown in FIG. 1 (a), the MoS obtained in the comparative example 2 The surface of the nano-grain structure is provided with ravines, the stacked thin nano-layers are in a fold shape, and the appearance of the popcorn sphere shows that the surface area of the nano-grain structure is large. As shown in FIG. 1 (b), in combination with a high-power transmission electron microscope (HR-TEM) and its corresponding Fourier Transform (FT), it can be observed that the MoS obtained by the comparative example preparation 2 Has a clear arrangement of lattice fringes on the surface, indicating its existenceHas good crystallinity, and is notable that the surface has obvious defect vacancies as shown by circles in FIG. 1 (b), and the lattice spacing of the surface is 0.256nm, which is MoS 2 The FT image shows the MoS obtained by the comparative example 2 Is polycrystalline. SAED image of comparative example As in FIG. 1 (c), the MoS obtained by the comparative example was further verified 2 Is polycrystal or MoS 2 A (101) plane and a (106) plane. Comparative example MoS obtained 2 HAADF-STEM image and corresponding energy spectrum analysis (EDS) element mapping image, as shown in fig. 1 (d), demonstrating MoS 2 The Mo element and the S element are uniformly distributed in the nanometer flower sphere structure.
The monoatomic nanoenzyme Pt@MoS prepared in example 1 was observed by using a Transmission Electron Microscope (TEM) 2 The microscopic surface morphology of (2) is shown in e-h of FIG. 1. As shown in FIG. 1 (e), the monoatomic nano-enzyme Pt@MoS can be observed 2 MoS obtained by comparison with comparative example 2 Also has a nanometer flower ball structure. HR-TEM and FT images as in FIG. 1 (f) can be observed to have a clear lattice fringe alignment after surface ion implantation, indicating good crystallinity, and the surface ion implantation technique does not alter the basic morphology, the surface lattice spacing of 0.256nm and 0.258nm, referred to as MoS 2 The surface of (102) has a significant defect as indicated by a circle in fig. 1 (f). The SAED image of example 1 is shown in FIG. 1 (g), further verifying that the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 Is polycrystal and is a base material MoS 2 A (112) plane and a (-102) plane. Example 1 Single atom nano enzyme Pt@MoS prepared 2 The HAADF-STEM image and the EDS element mapping image of the formula (I) are shown in (h) of figure 1, and the successful load of Pt element is proved, and Mo element, S element and Pt element are uniformly distributed in the nano flower sphere structure. Proved that the high-energy metal ion implantation technology can realize the successful loading of the metal element Pt without changing the MoS of the substrate material 2 The overall morphology and structure of (a).
Observation by a double-spherical-aberration correcting electron microscope is adopted to confirm that the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 The monoatomic sites of (a) to (d) are shown in FIG. 2. As shown in (a) of fig. 2,the HAADF-STEM image under the spherical aberration microscope can be observed to be obviously distributed in the monoatomic nano enzyme Pt@MoS prepared in example 1 2 The Pt monoatomic metal site can be clearly seen at the atomic scale by locally re-enlarging the metallic bright spot in (a) as shown in fig. 2 (b). As shown in FIG. 2 (b, c), the monoatomic nanoenzyme Pt@MoS prepared in example 1 was obtained at different viewing angles 2 The atomic scale Pt atomic scale metal sites were also observed in the HAADF-STEM image of (A), and these distinct bright spots were dispersed in the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 In the nanoflower structure of (2), moS can be observed as in (d) of FIG. 2 2 The above results demonstrate that the high energy ion implantation technique can achieve successful loading of Pt monoatoms.
The local coordination of Pt foil and Pt monoatoms of example 1 was studied using X-ray absorption spectroscopy (XAS) test, as shown in fig. 3 (a-b). XPS test was performed on the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 The chemical structure characterization of Pt 4f of (c) is shown in fig. 3. FIG. 3 (a) shows Pt foil and the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 Pt L of (2) 3 The XANES spectrogram is characterized in that the monoatomic nano-enzyme Pt@MoS prepared in example 1 is in the front peak area of the edge 2 The peak energy is higher than Pt foil. As shown in FIG. 3 (b), for Pt foil and the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 Performing Fu Libian leaf transformation under R space to obtain EXAFS spectrum, and performing Pt foil at radial distance
The position of (2) has obvious characteristic peak, which is named as Pt-Pt shell layer, and the monoatomic nano enzyme Pt@MoS prepared in example 1 2 At a radial distance of about->
There is a distinct Pt-Mo characteristic peak w position at +.>
The Pt-Pt characteristic peak position is not detected, and the result of combining with a spherical aberration electron microscope further proves thatPlatinum Metal atoms supported on MoS as monoatomic Pt 2 . FIG. 3 (c) shows the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 The peak of the Pt 4f XPS spectrum of (C) is found to be located at about 72.5eV (Pt 4f 7/2 ) And 75.8eV (Pt 4 f) 5/2 ) Bimodal composition, monoatomic nanoenzyme Pt@MoS prepared in example 1 2 The result of the deconvolution of XPS spectrum peaks shows that the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 The Pt species was oxidized and peak positions at 72.3eV and 73.1eV correspond to Pt 4+ Peak position at 75.8eV is ascribed to Pt 2+ Peak position of 76.5eV is ascribed to Pt + This is due to Pt monoatoms and MoS 2 Electronic interactions between them.
Preparing a test solution: mixing the sample with water to obtain a test solution with a sample concentration of 1mg/mL, wherein the sample is the monoatomic nano enzyme Pt@MoS prepared in the examples 1-3 2 And comparative example preparation of the obtained MoS 2 One of them.
mu.L of the test solution, 11. Mu.L of 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO, 500mM,McLean), 49. Mu.L of phosphate buffer (PBS, pH=7.4, hyClone) and 70. Mu. L H were mixed 2 O 2 Uniformly mixing to obtain a first reaction system;
mixing 70 μl of the test solution, 11 μl of 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO, 500mM,McLean) and 49 μl of phosphate buffer (PBS, ph=7.4, hyclone) uniformly to obtain a second reaction system;
a proper amount of the first reaction system or the second reaction system is sucked by using a capillary glass tube, the capillary glass tube is sealed by silicone grease, the capillary glass tube is cleaned and then placed into a paramagnetic tube, an electron spin resonance spectrometer (EMXplus-6/1, bruker, germany) is adopted for verifying hydroxyl radicals at room temperature, the microwave operating frequency is X-band, 9.8GHz, and the experimental result is shown in figure 4. FIG. 4 shows, from left to right, comparative examples and examples 3-1 under a DMPO capture agent (with or without H 2 O 2 ) Wherein the upper curve of the ESR spectrum is the second reaction system and the lower curve of the ESR spectrum is the first reaction system. It can be seen from FIG. 4 that the sample does not contain H 2 O 2 In the case of the second reaction system of (2)The ratio and spectral lines of examples 1-3 are baseline, without distinct characteristic signals, but at the presence of H 2 O 2 The spectral lines of comparative examples and examples 1 to 3 show a distinct characteristic signal in the case of the first reaction system of (1) can be clearly observed: 2:2:1 hydroxyl radical (-OH) profile, and the peak intensities were ordered as: example 1>Example 2>Example 3>Comparative example. The results of the spectra show that example 1 is superior to other examples in that it can release a large amount of toxic-OH, and the high level of Reactive Oxygen Species (ROS) can cause oxidative stress of cancer cells under specific tumor microenvironment (TEM), inducing hypoxia-toxicity death of tumor cells. Plays a vital role in inhibiting the proliferation of cancer cells.
Evaluation of the MoS obtained in the comparative example using 4T1 cells (Tianjin New technology Co., ltd.) 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Is effective in inhibiting proliferation of cancer cells.
Culturing 4T1 cells (4T 1 cell culture Environment: 95% air, 5% CO) 2 A constant temperature incubator at 37 ℃. The cell complete medium was changed once daily and passaged once for two days. ) Taking 4T1 cells in logarithmic growth phase. Sucking off complete cell culture medium, adding 5mL PBS (pH=7.4) to rinse 4T1 cells, sucking off the PBS, adding 1mL 0.25% pancreatin-0.02% EDTA (BI), placing into a constant temperature incubator at 37deg.C for digestion, adding 6mL complete cell culture medium to terminate digestion, gently blowing off adherent cells, mixing, centrifuging the mixed cells in a centrifuge for 3min, sucking off supernatant, adding 5mL complete cell culture medium, mixing, and finally obtaining the final product with inoculation density of 1×10 4 cell suspension in cell/mL, 1mL of cell suspension was added to each well of a 24-well plate, incubated in a constant temperature incubator at 37℃for 24 hours, and the supernatant was removed by vacuum pump.
MoS obtained by comparative example 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 Sterilizing under ultra-clean bench ultraviolet lamp for 24 hr, and collecting MoS obtained in comparative example 2 And the monoatomic nanoenzyme Pt@MoS prepared in examples 1-3 2 The cell complete medium was diluted to c. Mu.g/mL to give a material liquid, c. Mu.g/mL was 50. Mu.g/mL, 100. Mu.g/mL or 200. Mu.g/mL, respectively.
1mL of the material liquid was added to the wells filled with cells in the 24-well plate as experimental wells. 1mL of cell complete medium was added to wells in the 24-well plate in which cells were loaded as control wells. The blank wells were used as 24-well plate wells (no cells).
The 24-well plates of the experimental and control wells were placed in a 37 ℃ incubator for incubation t h, t h for 0, 2h,6h,12h or 24h. After the incubation was completed, the supernatant was aspirated, and washed three times for 5min each with PBS.
To each of the experimental, control and blank wells, 270. Mu.L of RPMI-1640 serum-free medium (BIOIND) and 10. Mu.L of a standard cell counting kit (CCK-8, genview) were added as a mix for cytotoxicity detection. After 2 hours incubation in a constant temperature incubator at 37℃in the absence of light, a detection mixture was obtained, and 300. Mu.L of the detection mixture was transferred to a 96-well plate with 100. Mu.L of each well. The cell viability of the blank wells was defined as 0% and the cell viability of the control wells as 100%, the Optical Density (OD) of the wells was recorded using an enzyme-labeled instrument (ELX 808IU, bioTek, USA) at a wavelength of 450nm and the experimental results are shown in fig. 5a and b.
FIG. 5a is a graph showing the change in the inhibition rate of 4T1 cell proliferation at T h of 0h,2h,6h,12h or 24h for the comparative example (comparative example in FIG. 5) and examples 1 to 3 c. Mu.g/mL=200. Mu.g/mL, and it can be observed that the monoatomic nanoenzyme Pt@MoS prepared in example 1 under different action times 2 The 4T1 cell proliferation inhibition ability of (2) was strongest, the proliferation inhibition change rates of comparative examples and examples 1 to 3 tended to be smooth at 12h, the cell survival rate of example 1 was only 8.9% at 24h, and the proliferation inhibition rate for 4T1 was as high as 91.1%.
FIG. 5 b is a graph showing that the comparative examples and examples 1 to 3 show significantly better cancer cytotoxicity than the comparative examples at a low concentration of 50. Mu.g/mL, showing a change in cell proliferation inhibition rate of 24 hours for 4T1 cells at a concentration of c. Mu.g/mL, 100. Mu.g/mL or 200. Mu.g/mL, showing that the Pt-loaded examples 1 to 3 on the surface of the ion implantation have a stronger proliferation inhibition ability than the comparative examples, indicating that the ion implantation modified MoS 2 The proliferation inhibition capability of cancer cells is enhanced, and the proliferation inhibition capability of 4T1 cells is still enhanced at low concentration, when the concentration is 10The inhibition rate of change of proliferation of 4T1 cells of example 1 at 0 μg/mL and 200 μg/mL was smoothed, indicating that the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 The Pt monoatomic site of (2) has strong inhibition effect on cancer cell proliferation, and the monoatomic site effectively catalyzes the oxidation-reduction reaction of 4T1 cells under a specific TEM, and the monoatomic nano-enzyme Pt@MoS prepared in the embodiment 1 2 Can produce large amounts of toxic-OH, and high levels of ROS lead to oxidative stress and hypoxia apoptosis of 4T 1.
Calcein-AM and PI (Beyotidme) were co-stained with 4T1 cells to detect 4T1 cell activity and cytotoxicity. The detection method comprises the following steps: taking 4T1 cells in logarithmic growth phase, inoculating onto confocal special cell culture dish (Biosharp) with cell complete culture medium, inoculating density of 3×10 4 cell/mL, incubated for 24h in a constant temperature incubator at 37℃and then supernatant was aspirated and PBS (pH=7.4 as no treatment in FIG. 6) or 1mL of a cell complete medium mixture (as comparative example in FIG. 6, examples 1 to 3) was added to act for 12h, after which 4T1 cells were washed with PBS (pH=7.4) to obtain cells to be stained, wherein the cell complete medium mixture was a mixture of cell complete medium and material, and the material was MoS prepared in comparative example 2 (comparative example in FIG. 6) and the monoatomic nanoenzyme Pt@MoS prepared in examples 1 to 3 2 The concentration of the material in the cell complete medium mixture was 100. Mu.g/mL.
According to the instructions of the Biyun cell Activity and cytotoxicity assay kit (C2015M, shanghai Biyun Biotechnology Co., ltd.), 0.3. Mu.L of Calcein-AM, 0.3. Mu.L of PI and 3. Mu.L of assay buffer were mixed to obtain a mixture, which was added to the cells to be stained at 300. Mu.L per well and incubated at 37℃for 40min in the absence of light. After the completion of the incubation in the dark, the living and dead state of 4T1 cells was observed under a laser confocal microscope (FV 1000, olympus, japan), and Calcein-AM was green fluorescent (Ex/Em=488/517 nm) and PI was red fluorescent (Ex/Em=546/617 nm), and the experimental results are shown in FIG. 6.
FIG. 6 is a laser co-dye microscope image of Calcein-AM and PI for 4T1 cells of comparative and examples 1-3. The best adherent growth state of 4T1 cells in the non-treated group and no dead cells can be observed. Under the action of comparative examples and examples 1 to 3, 4T1 cells were killed, and the number of 4T1 cells was gradually increased by red fluorescence (toxicity inhibition strength: example 1>Example 2>Example 3>Comparative example) in which the monoatomic nanoenzyme Pt@MoS prepared in example 1 2 The inhibition effect on 4T1 cells is strongest, the number of green fluorescent 4T1 cells is obviously reduced, the morphology of 4T1 cells is obviously destroyed, most 4T1 cells are in a single state, most 4T1 cells are in a floating state and can not be adhered to the wall, even some 4T1 cells are crushed, which indicates that the 4T1 cells are subjected to the preparation of the monoatomic nano enzyme Pt@MoS in the example 1 2 Is basically unable to survive under the action of the cell line, 4T1 cells shrink into a sphere shape, and cell membranes are destroyed, so that a large number of 4T1 cells apoptosis. This is consistent with the result of FIG. 5, which shows that the monoatomic nano-enzyme Pt@MoS prepared in example 1 2 Has obvious toxicity inhibiting effect on 4T1 cell proliferation.
The above comprehensive description shows that the high-energy ion implantation technology can realize the effective load of the monoatomic Pt, and the monoatomic nano-enzyme Pt@MoS prepared by the invention 2 Has a prospect in effectively utilizing the monoatomic active site to simulate the natural enzyme to inhibit the proliferation direction of cancer cells.
This patent was funded by national natural science foundation (project number: 51772209) and by the Tianjin master university scientific research innovation project (project number: 2022KYCX 024Z).
The foregoing has described exemplary embodiments of the invention, it being understood that any simple variations, modifications, or other equivalent arrangements which would not unduly obscure the invention may be made by those skilled in the art without departing from the spirit of the invention.
Claims (9)
1. Monoatomic nano-enzyme Pt@MoS 2 The preparation method of (2) is characterized by comprising the following steps:
1) Mixing potassium thiocyanate and ammonium molybdate tetrahydrate, preserving heat for 1.5-2.5 hours at the temperature of 250-350 ℃ to obtain black solid, washing the black solid with water, and drying to obtain MoS 2 Wherein, the ratio of potassium thiocyanate to ammonium molybdate tetrahydrate is (3-6) according to the mass parts: (0.1 to 0.2);
2) MoS is carried out 2 Uniformly mixing the mixture with a solvent to obtain a dispersion, wherein the solvent is a mixture of absolute ethyl alcohol and deionized water, the ratio of the absolute ethyl alcohol to the deionized water is 1 (3-4) in parts by volume, and MoS in the dispersion is obtained 2 The concentration of (2) is 0.006-0.0075 g/mL;
3) Dripping the dispersion liquid on a conductive carrier, and drying to obtain a substrate, wherein MoS is dripped into the dispersion liquid per square centimeter of the conductive carrier 2 The mass of (2) is 0.0025-0.005 g;
4) Injecting Pt metal plasma into the surface of the substrate, on which the dispersion liquid is dripped, for 80-150 min to obtain the monoatomic nano enzyme Pt@MoS 2 The high-energy ion implanter is used for implanting Pt metal plasma, the high-voltage pulse of the cathode of the high-energy ion implanter is triggered to lead out the Pt metal plasma from the anode Pt target, and the vacuum degree in a chamber for implanting the Pt metal plasma is lower than 6 multiplied by 10 -3 pa。
2. The method according to claim 1, wherein in the step 1), the drying temperature is 60 to 80 ℃ and the drying time is 18 to 24 hours.
3. The method according to claim 1, wherein in the step 1), after mixing potassium thiocyanate and ammonium molybdate tetrahydrate, the temperature is raised from room temperature of 20 to 25 ℃ to the temperature of 250 to 350 ℃ at a rate of 5 to 10 ℃/min.
4. The method of claim 1, wherein in step 3), the conductive support is a metal sheet or a conductive glass sheet.
5. The method according to claim 4, wherein in the step 3), the drying temperature is 60 to 80 ℃ and the drying time is 10 to 12 hours.
6. The method of claim 1, wherein in step 4) the high energy ion implanter is a MEVVA type source.
7. The method according to claim 1, wherein in the step 4), the trigger frequency of the cathode high voltage pulse is 8-15 Hz, the trigger voltage is 3-6 kV, the arc voltage is 40-150V, the arc current is 0.4-1.5A, the suppression voltage is 0.6-1.1 kV, the injection voltage is 10-50 kV, and the extraction current is 0.2-0.5A.
8. The method according to claim 1, wherein in the step 4), after the injection of Pt metal plasma is completed, the substrate is washed with absolute ethanol by: immersing the substrate in absolute ethyl alcohol, performing ultrasonic treatment for 1.5-2 hours, performing vacuum filtration, and drying the solid obtained by vacuum filtration at 60-80 ℃ for 10-12 hours.
9. The monoatomic nano-enzyme Pt@MoS obtained by the preparation method according to any one of claims 1-8 2 。
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