Iron-doped molybdenum disulfide nano material as well as preparation method and biological application thereof

Abstract

The invention belongs to the field of nano materials, and particularly relates to an iron-doped molybdenum disulfide nano material as well as a preparation method and biological application thereof. The iron-doped molybdenum disulfide nano material has a sharp edge, a photothermal conversion function and peroxidase catalytic activity, provides a mechanical effect, a thermal effect and an active oxygen effect on damage of biological membranes and bacteria, has good sterilization and anti-biological membrane activity, and can be used for preparing peroxidase-like materials and materials with sterilization activity. The peroxidase-like catalytic activity of the iron-doped molybdenum disulfide nano-material can be adjusted through DNA modification, and the iron-doped molybdenum disulfide nano-material is used for protein detection. The iron-doped molybdenum disulfide nano material is diversified in function, simple to prepare, low in cost and wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and specifically relates to a multifunctional iron-doped molybdenum disulfide nanomaterial and a preparation method and biological application thereof. Background Art

[0002] Proteins are important components of living organisms and are involved in almost every process within the cell, which performs a wide range of functions within the organism, including catalyzing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. For example, enzymes that catalyze biochemical reactions have the function of promoting metabolism. Membrane proteins play a special role in structure and function. There is a great demand for rapid, low-cost detection and identification of proteins in the biomedical and food industries. In recent decades, the need for reliable and accurate sensing has driven the development of various protein detection methods, each with its own advantages and disadvantages. For example, immunoblotting is a highly sensitive and specific detection method that works by exploiting the inherent specificity of antigen-antibody recognition. However, it is costly and requires precise technology. Enzyme-linked immunosorbent assay is a powerful tool for high-sensitivity protein detection, but its disadvantages include low specificity, considerable turnaround time, and complex labeling process. The sensor array selects a variety of sensor units to combine together, has no specific binding to proteins, breaks the traditional lock-and-key mode, has high sensitivity, is not affected by environmental factors and operators during the detection process, has strong anti-interference ability, and can complete the detection of a large number of samples in a short time, improving the detection efficiency. And for the construction of the sensor unit, we only need to modify the material with different lengths of PolyDNA to obtain different enzyme catalytic properties, which greatly saves time and cost.

[0003] Dental caries is a global risk for the oral health of many school-age children and the vast majority of adults. The main factor in the formation of dental caries is the attachment of bacteria to the tooth surface to form oral biofilms, which produce acid decalcification and ultimately lead to tooth decay. Some microorganisms such as Streptococcus mutans are acid-resistant and acid-producing, and therefore, S. mutans plays an important role in the occurrence and development of dental caries. For this reason, the elimination of oral biofilms remains a challenge because the bacteria are embedded in them and the biofilm matrix protects them, blocking the penetration of antibiotics or bactericides. Summary of the invention

[0004] The Fe-MoS2 nanomaterial provided by the present invention is a multifunctional material that can simulate the function of natural peroxidase, catalyze H2O2 to produce highly toxic reactive oxygen species (ROS) as an antibacterial substance, and in addition, has a high photothermal conversion efficiency in the near-infrared region. Therefore, by using the dual functions of simulating natural enzymes and photothermal conversion, the Fe-MoS2 nanomaterial can achieve the effect of synergistic sterilization and anti-biofilm. At the same time, the Fe-MoS2 nanomaterial provided by the present invention also has a good mechanical damage function in sterilization and anti-biofilm. The iron-doped molybdenum disulfide nanomaterial provided by the present invention can adjust its peroxidase-like catalytic activity through DNA modification, thereby realizing protein detection.

[0005] The invention provides an iron-doped molybdenum disulfide nanomaterial (Fe-MoS2), which is in a dendrite shape with sharp edges and in which the three elements of iron, molybdenum and sulfur are evenly distributed.

[0006] The atomic proportions of the three elements Fe, Mo and S are 9.1%, 42.94% and 47.96% respectively.

[0007] Furthermore, the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention is also modified with DNA, wherein the DNA is preferably ssDNA, and more preferably PolyDNA, and the sequence of the PolyDNA is preferably:

[0008] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0009] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0010] Sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.

[0011] The preparation method of the above-mentioned iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention comprises the following steps: adding potassium ferrocyanide and ammonium tetrathiomolybdate into a DMF solution and mixing evenly, and performing heat treatment in a hydrothermal reactor.

[0012] The mass ratio of potassium ferrocyanide to molybdenum tetrathioate is 1:1-3, preferably 1:2-2.5, more preferably 1:2, and the concentration of molybdenum tetrathioate is 1-2 g / L, preferably 1.5-1.7 g / L.

[0013] Heat treatment at 200°C-300°C for 18-24 hours, preferably heat treatment at 200°C for 24 hours.

[0014] After the heat treatment, the mixture was separated, washed, dried, centrifuged at 9000 rpm / min, washed three times with deionized water and anhydrous ethanol respectively, and dried under vacuum at 50° C. for 12 hours.

[0015] The invention provides a method for preparing iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) modified with DNA, the steps comprising: incubating the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) and DNA in a fixed buffer.

[0016] The fixing buffer is: 10 mM HAC-NaAC, pH=4.0.

[0017] The DNA is preferably ssDNA, and more preferably PolyDNA, and the sequence of PolyDNA is preferably:

[0018] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0019] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0020] Sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.

[0021] The iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention has a catalytic activity similar to that of peroxidase, catalyzes the redox reaction of a substrate (color indicator, such as TMB) using hydrogen peroxide as an electron acceptor, and can be used to prepare peroxidase-like enzymes or peroxidase mimetic enzymes.

[0022] The iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention can adsorb and modify different DNAs (such as ssDNA), and different DNAs (such as ssDNA) can adsorb and polymerize different proteins. The charge, size and hydrophobicity of different proteins are different, so that the outermost layer of the iron-doped molybdenum disulfide nanomaterial exhibits different binding affinities, and the binding affinity between the iron-doped molybdenum disulfide nanomaterial and the substrate changes to varying degrees, causing the regulation of the peroxidase-like activity of the iron-doped molybdenum disulfide nanomaterial and different colorimetric reactions, thereby achieving accurate identification of proteins. Therefore, the iron-doped molybdenum disulfide nanomaterial that adsorbs and modifies different DNAs (such as ssDNA) provided by the present invention has adjustable peroxidase-like catalytic activity and can be used to prepare peroxidase-like enzymes or peroxidase mimetic enzymes. The iron-doped molybdenum disulfide nanomaterial and the iron-doped molybdenum disulfide nanomaterial that modifies different DNAs (such as ssDNA) provided by the present invention can be used to prepare materials or sensing units or sensors for protein detection.

[0023] The iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention has a good photothermal conversion effect, converting the absorbed light energy into heat energy, so that the temperature of the iron-doped molybdenum disulfide material and its surrounding environment rises rapidly, and the high temperature destroys the bacterial cell membrane structure; at the same time, it has peroxidase-like activity, catalyzing H2O2 to produce reactive oxygen species that are highly toxic to bacteria, which destroys cell membranes, damages DNA, and especially cell necrosis; the sharp edges of its dendritic structure produce strong mechanical and physical damage to biofilms and bacterial cell membranes; the above-mentioned effects synergistically and effectively destroy biofilms and kill bacteria, showing excellent bactericidal activity. Therefore, the present invention provides iron-doped molybdenum disulfide nanomaterials and iron-doped molybdenum disulfide nanomaterials modified with different ssDNAs that can be used to prepare medical, sanitary materials or auxiliary materials, and the material has bactericidal activity. Furthermore, the present invention provides iron-doped molybdenum disulfide nanomaterials and iron-doped molybdenum disulfide nanomaterials modified with different ssDNAs, which can be used to prepare materials or auxiliary materials for teeth and periodontal tissues, and the materials have bactericidal activity; further, the present invention provides iron-doped molybdenum disulfide nanomaterials and iron-doped molybdenum disulfide nanomaterials modified with different ssDNAs, which can be prepared into filling materials or auxiliary filling materials for defective or carious teeth and periodontal tissues, and the filling materials have bactericidal activity.

[0024] The present invention provides a peroxidase-like enzyme or a peroxidase mimetic enzyme, which contains the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention and / or the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) for modifying DNA provided by the present invention.

[0025] Furthermore, the DNA is preferably ssDNA, and further preferably PolyDNA, and the sequence of PolyDNA preferably includes:

[0026] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0027] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0028] Sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.

[0029] The present invention provides a material or a sensing unit or a sensor for protein detection, which contains the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) provided by the present invention and / or the iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) for modifying DNA provided by the present invention.

[0030] Furthermore, the sensor comprises a sensor array formed by a combination of several sensor units. The sensor units comprise iron-doped molybdenum disulfide nanomaterials for modifying DNA, and different sensor units modify different DNA.

[0031] Furthermore, the DNA is preferably ssDNA, and further preferably PolyDNA, and the sequence of PolyDNA preferably includes:

[0032] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0033] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0034] Sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.

[0035] The present invention provides a method for detecting protein, comprising the following steps: incubating a sample to be tested, an iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) and DNA in a fixed buffer or incubating a sample to be tested and an iron-doped molybdenum disulfide nanomaterial modified with DNA in a fixed buffer, then adding H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) for incubation, measuring the absorbance value at 652nm, and performing qualitative and / or quantitative detection of the protein.

[0036] The present invention provides a method for detecting proteins with a sensor array, the steps comprising:

[0037] (1) placing the sample to be tested, iron-doped molybdenum disulfide nanomaterial (Fe-MoS2) and DNA in an incubation well with a fixed buffer added to a sensor unit constituting a sensor array for incubation, or placing the sample to be tested and iron-doped molybdenum disulfide nanomaterial modified with DNA in an incubation well with a fixed buffer added to a sensor unit constituting a sensor array for incubation, then adding H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) for incubation, and measuring the absorbance value at 652 nm;

[0038] (2) Perform data analysis on the absorbance value at 652 nm and perform qualitative and / or quantitative detection of single target protein and mixed protein.

[0039] In step (1), the fixing buffer is: 10 mM HAC-NaAC, pH = 4.0.

[0040] In step (1), the sensing units are different and the modified DNA is different; the DNA is preferably ssDNA, and more preferably PolyDNA, and the sequence of PolyDNA is preferably:

[0041] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0042] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0043] The sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC;

[0044] Specifically, PolyC6 is modified to generate a PolyC6 / Fe-MoS2 sensing unit, PolyC15 is modified to generate a PolyC15 / Fe-MoS2 sensing unit, and PolyC45 is modified to generate a PolyC45 / Fe-MoS2 sensing unit.

[0045] In step (1), the sensing unit may be a well plate for biological detection.

[0046] In step (1), the sample to be tested is first pre-processed.

[0047] In step (2), the data analysis is PCA principal component analysis, and the distribution range of the protein is determined based on the 95% confidence ellipse in the PCA analysis graph.

[0048] In step (2), the single target protein comprises bovine hemoglobin (BHB), concanavalin A (ConA), cytochrome C (CytC), human serum albumin (HSA), chymotrypsinogen A (ChyA), pepsin (Pep), papain (Pap), and proteinase K (Pro K), and the mixed protein is a binary mixture of cytochrome C and chymotrypsinogen A.

[0049] The present invention provides a medical or sanitary material or auxiliary material, or a material or auxiliary material for teeth and periodontal tissues, or a filling material or auxiliary filling material for teeth or periodontal tissues, which contains the iron-doped molybdenum disulfide nanomaterial provided by the present invention and / or the iron-doped molybdenum disulfide nanomaterial for modifying DNA provided by the present invention.

[0050] Furthermore, the DNA is preferably ssDNA, and further preferably PolyDNA, and the sequence of PolyDNA preferably includes:

[0051] PolyC6 sequence (SEQ ID NO: 1): CCCCCC; and / or,

[0052] Sequence of PolyC15 (SEQ ID NO: 2): CCCCCCCCCCCCCCC; and / or,

[0053] Sequence of PolyC45 (SEQ ID NO: 3): CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.

[0054] Compared with the prior art, the advantages of the present invention are:

[0055] (1) In the detection of proteins, the sensor array selects a variety of sensing units to be combined together, which has no specific binding to proteins, breaking the traditional lock-key mode, has high sensitivity, is not affected by environmental factors and operators during the detection process, has strong anti-interference ability, and can complete the detection of a large number of samples in a short time, improving the detection efficiency. And for the construction of the sensing unit, we only need to modify the material with PolyDNA of different lengths to obtain different enzyme catalytic properties, which greatly saves time and cost.

[0056] (2) In terms of antibacterial and anti-biofilm, Streptococcus mutans, the main microorganism that causes dental caries, has acid production and acid resistance. Fe-MoS2 nanomaterials, as a multifunctional material, can simulate the function of natural peroxidase and have high enzyme catalytic activity under acidic conditions. They can catalyze H2O2 to produce highly toxic reactive oxygen species (ROS) as antibacterial substances. In addition, they have high photothermal conversion efficiency in the near-infrared region. Therefore, by utilizing the dual functions of simulating natural enzymes and photothermal conversion, Fe-MoS2 nanomaterials can achieve synergistic bactericidal and anti-biofilm effects. For teeth that have already formed substantial defects, the carious tissue is removed and a certain cavity shape is made before filling. Commonly used materials include glass ionomers, light-cured resins, etc., and Fe-MoS2 is expected to be used alone as a filling material or auxiliary filling material to treat dental caries.

[0057] (3) In addition, Fe-MoS2 nanomaterials are simple to synthesize, low-cost, and can be prepared in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Shown is a schematic diagram of an overall solution provided by an embodiment of the present invention.

[0059] Figure 2 Characterization results of Fe-MoS2, a: TEM image; b: EDS-Mapping element analysis diagram, green is iron, red is molybdenum, and blue is sulfur; c: SEM image; d: XRD spectrum, the upper red line is MoS2, and the lower orange line is Fe-MoS2.

[0060] Figure 3 The following are the UV spectra of Fe-MoS2 peroxidase modified by three types of PolyDNA (C6, C15, C45). Fe-MoS2 modified by different chain length DNA showed different enzyme catalytic activities, C45>C15>C6≈Fe-MoS2 without DNA modification. This shows that the three types of DNA have different effects on Fe-MoS2.

[0061] Figure 4 This is a graph showing the protein differentiation results of the material detection sensor array composed of three nanozymes: PolyC6 / Fe-MoS2, PolyC15 / Fe-MoS2 and PolyC45 / Fe-MoS2.

[0062] Figure 5 Material detection sensor array composed of three nanozymes: PolyC6 / Fe-MoS2, PolyC15 / Fe-MoS2 and PolyC45 / Fe-MoS2. a: Single CytC concentration gradient result diagram and b: Linear relationship between CytC concentration and the first discriminant factor PC1.

[0063] Figure 6 This is a graph showing the results of the material detection sensor array composed of three nanozymes, PolyC6 / Fe-MoS2, PolyC15 / Fe-MoS2 and PolyC45 / Fe-MoS2, detecting the binary mixture of CytC and ChyA.

[0064] Figure 7 This is a diagram showing the inhibitory effect of Fe-MoS2 on Streptococcus mutans.

[0065] Figure 8 This is a diagram showing the inhibitory effect of Fe-MoS2 on Streptococcus mutans biofilm. DETAILED DESCRIPTION

[0066] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0067] Figure 1 Schematic diagram of the overall solution adopted in the embodiment of the present invention, a: protein detection schematic diagram, b: antibacterial and anti-biofilm schematic diagram.

[0068] Example 1 Synthesis of Fe-MoS2

[0069] Fe-MoS2 was prepared by solvothermal method. Specifically, 25 mg potassium ferrocyanide and 50 mg ammonium tetrathiomolybdate were weighed and added to 30 mL DMF solution, transferred to a 50 mL hydrothermal reactor after ultrasonic treatment and placed in an oven at 200 ° C for 24 hours. After cooling to room temperature, centrifuged at 9000 rpm / min for 2 minutes, washed three times with deionized water and anhydrous ethanol respectively to obtain a black precipitate, which was then placed in a 50 ° C vacuum drying oven and dried for 12 hours to obtain a black solid.

[0070] The obtained Fe-MoS2 SEM, TEM, Mapping, and XRD are as follows Figure 2 As shown, through Figure 2 a TEM image and Figure 2 c SEM image shows that after Fe doping of MoS2, a dendritic structure with sharp edges is formed, which provides the possibility of inducing destruction of bacterial cell membranes through mechanical physical damage. Figure 2 b is the elemental analysis (EDS-Mapping) of Fe-MoS2. It can be seen that the three elements Fe, Mo and S are evenly distributed, and the atomic proportions of Fe, Mo and S are 9.1%, 42.94% and 47.96% respectively. Figure 2d is the XRD spectrum of MoS2 and Fe-MoS2. After comparing with the standard card, it can be seen that there is no obvious heterojunction peak after the incorporation of Fe, indicating that Fe mainly exists in the form of doping. These results further illustrate that Fe-MoS2 was successfully synthesized.

[0071] Example 2 Verification of the activity of three DNA-modified Fe-MoS2-type peroxidase mimics

[0072] 3 μL of 200 μg / mL Fe-MoS2 and 15 μL of 10 μM PolyDNA (C6, C15, C45) were added to 276 μL of HAC-NaAC buffer (10 mM, pH 4.0), mixed and allowed to stand at room temperature for 20 minutes, then 3 μL of 50 mM H2O2 and 3 μL of 10 mM TMB were added, incubated at room temperature for 10 minutes, and the spectrum of the supernatant at 400-800 nm was immediately recorded on a UV spectrophotometer. The results are shown in FIG. Figure 3 As shown, the three DNA-modified nanozymes all exhibited obvious absorption peaks at 652 nm.

[0073] Wherein, the sequence of PolyC6 is: CCCCCC; (SEQ ID NO: 1)

[0074] The sequence of PolyC15 is: CCCCCCCCCCCCCCC; (SEQ ID NO: 2)

[0075] The sequence of PolyC45 is: CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

[0076] CCCCCCCCCCCCCCC. (SEQ ID NO:3)

[0077] Example 3 A material detection sensor array composed of three kinds of DNA and Fe-MoS2 nanozymes distinguishes proteins, comprising the following steps:

[0078] (1) Determine at least one sample to be tested and prepare the sample to be tested, including: weighing different samples to be tested and placing them in test tubes respectively, and then adding deionized water to dissolve them to obtain a protein solution with a concentration of 1 mg / mL.

[0079] (2) First, 178 μL (10 mM, pH = 4) of HAC-NaAC buffer was added to a 96-well plate, followed by 2 μL of 100 μg / mL Fe-MoS2. Next, 6 μL of 10 μM PolyC6, PolyC15, and PolyC45 were added to the wells of the corresponding well plate rows and incubated at room temperature for 20 minutes. Then, 10 μL of 1 mg / mL of the test protein was added to the wells containing PolyC6 / Fe-MoS2, PolyC15 / Fe-MoS2, and PolyC45 / Fe-MoS2 DNA-modified nanozymes, respectively. After mixing, the wells were incubated at room temperature for 30 minutes. After the incubation, 2 μL of 50 mM H2O2 solution and 2 μL of 20 mM 3,3',5,5'-tetramethylbenzidine solution (TMB) were added in sequence. After another 10-minute incubation, the absorbance value data was measured at 652 nm using an ELISA reader. Repeat the above steps for each protein to be tested.

[0080] (3) After the measured absorbance values ​​were subjected to PCA principal component analysis, single target proteins and mixed proteins were obtained respectively. The single target proteins included: bovine hemoglobin from blood (BHB), concanavalin A (ConA), cytochrome C (CytC), human serum albumin (HSA), chymotrypsinogen A (ChyA), pepsin (Pep), papain (Pap), proteinase K (Pro K). The mixed protein was a binary mixture of CytC and ChyA.

[0081] (4) Test results Figure 4 As shown in the PCA map, through the coverage area of ​​the 95% confidence ellipse of each protein, it can be seen that the protein sensor array can distinguish 8 single protein arrays; secondly, the detection results are as follows Figure 5 As shown in the PCA graph, the 95% confidence ellipse coverage area of ​​different concentrations of CytC shows that the protein sensor array can distinguish different concentrations of CytC, with a detection limit as low as 2.24 μg / mL. Figure 6 As shown, in the PCA map, through the 95% confidence ellipse coverage area of ​​proteins with different mixing ratios, it can be seen that the protein sensor array can distinguish mixed proteins with different mixing ratios.

[0082] Principle of protein detection: ssDNA can be adsorbed on MoS2 through van der Waals force. PolyDNA (C6, C15, C45) of different lengths is selected to combine with Fe-MoS2 to construct three sensing units. The combination of PolyDNA (C6, C15, C45) of different lengths with Fe-MoS2 changes the catalytic performance of the material, and three sensing units are constructed. Due to the different charges, sizes and hydrophobicities of different proteins, they show different binding affinities to the different outermost interfaces of Fe-MoS2 nanozymes based on DNA adsorption. Therefore, it causes the binding affinity between the nanozyme of DNA-adsorbed Fe-MoS2 and the substrate to change to different degrees, thereby causing the regulation of peroxidase simulation activity and different colorimetric reactions, and the absorbance value is detected by an enzyme marker, and then after PCA analysis, the qualitative or quantitative analysis of the target protein can be quickly and conveniently achieved.

[0083] Example 4 Fe-MoS2 antibacterial experiment

[0084] (1) First, take a liquid of Streptococcus mutans in the logarithmic growth phase (about 10 8 CFU / mL) 1mL, centrifuge (10000rpm / min 5min), resuspend the precipitate in 1mL of normal saline, pipette and mix well for later use.

[0085] (2) Then, 20 μL of bacterial solution was added to the sterilized centrifuge tube, and control, H2O2, Fe-MoS2, and Fe-MoS2+H2O2 groups were set up. The Fe-MoS2 and H2O2 used were 50 μg / mL and 0.2 mM, respectively. Correspondingly, an illumination group was set up as a control, and the near-infrared light source (808 nm, 2 W / cm 2 ) for 8 minutes. Incubate in a microbial incubator at 37°C for 20 minutes. Then perform a gradient dilution (10 5 50 μL was plated (≥3 replicates per group), and then CFU counts were performed 48 hours later to calculate the bacterial survival rate.

[0086] (3) The bacterial survival rate was calculated using the following equation: A represents the negative control group without light and sample treatment, and B represents other groups except the negative control group.

[0087] (4) Figure 7As shown in the figure, when there is no light treatment, by calculating the bacterial survival rate, it can be obtained that the sterilization rate of the Fe-MoS2 treatment group can reach 89.7%, which may be due to the sharp edges of the dendritic Fe-MoS2 that can induce mechanical damage to the bacterial cell membrane, while the sterilization rate of the Fe-MoS2+H2O2 group can reach 97.1%, and after NIR (808nm, 2W / cm 2 , 8min) irradiation, the sterilization rate of both Fe-MoS2 and Fe-MoS2+H2O2 reached 100%, which shows that Fe-MoS2 has good antibacterial properties, in which light and heat play a major role.

[0088] Example 5 Fe-MoS2 anti-biofilm experiment

[0089] (1) In a clean bench, add 250 μL of BHI medium containing 1% sucrose and 225 μL of BHI medium to a sterilized 48-well plate; then take a culture of Streptococcus mutans in the logarithmic growth phase (about 10 8 CFU / mL), add 25μL to each well and place in a 37℃ microbial incubator for 24 hours. After 24 hours, the culture medium was aspirated and bacterial biofilm was formed at the bottom of the well plate. Add appropriate amount of physiological saline to each well and wash gently to remove residual suspended bacteria and culture medium.

[0090] (2) Control, H2O2, Fe-MoS2, and Fe-MoS2+H2O2 groups were set up, with Fe-MoS2 and H2O2 at 100 μg / mL and 0.2 mM, respectively. A light-exposed group was set up as a control, receiving a near-infrared light source (808 nm, 2 W / cm 2 ) for 8 minutes. Incubate in a microbial incubator at 37°C for 20 minutes. Then add 800 μL of saline to each well to rinse and suspend the biofilm fully. Pipette 1 mL of liquid in the well plate into a centrifuge tube and perform gradient dilution (10 dilution with saline). 5 50 μL was taken to plate, and CFU count was performed 48 hours later to calculate the bacterial survival rate.

[0091] (3) Figure 8 As shown in the figure, when there is no light treatment, the inhibition effect of Fe-MoS2 on biofilm is 23.2% by calculating the bacterial survival rate, while the inhibition rate of Fe-MoS2+H2O2 group is 64.1%. For the bacterial colonies that form biofilm, the effect of killing bacteria by physical and mechanical destruction of biofilm or active oxygen is not very satisfactory. However, after NIR (808nm, 2W / cm 2After irradiation for 8 minutes, the inhibition rate of the Fe-MoS2+H2O2 group can reach 90% due to the synergistic effect between them, which makes it possible for Fe-MoS2 to be used as a filling material for treating caries or as an auxiliary filling material.

[0092] Principle of sterilization and destruction of biofilm: MoS2 itself is a semiconductor material with good photothermal properties, and the material synthesized by iron doping has both good photothermal conversion effect and good peroxidase-like activity. Good photothermal conversion can convert the absorbed light energy into heat energy inside the material through a non-radiative relaxation process, causing the temperature of the material and its surrounding environment to rise rapidly. The high temperature generated will destroy the bacterial cell membrane structure, causing some components in the bacteria to leak out, and at the same time denature the proteins and enzymes in the bacteria, ultimately achieving the effect of killing bacteria. The nanozyme Fe-MoS2 can simulate natural peroxidase-like enzymes to catalyze H2O2 to produce reactive oxygen species (ROS). The formation of ROS is the main mechanism of nanoparticle sterilization, which can cause DNA damage, cell membrane damage and even cell necrosis. Therefore, through their synergistic effect, they can effectively sterilize and destroy biofilms.

[0093] Finally, it should be noted that the above description of the embodiments is for the convenience of understanding and use by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described below to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An iron-doped molybdenum disulfide nanomaterial, characterized in that: It is in the shape of a tree with sharp edges, and the three elements of iron, molybdenum and sulfur are evenly distributed.

2. An iron-doped molybdenum disulfide nanomaterial modified with DNA, characterized in that: The iron-doped molybdenum disulfide nanomaterial described in claim 1 is modified with DNA.

3. The method for preparing the iron-doped molybdenum disulfide nanomaterial according to claim 1, characterized in that: The steps include: Potassium ferrocyanide and ammonium tetrathiomolybdate are added into the DMF solution and mixed evenly, and then heat treated in a hydrothermal reactor.

4. The method for preparing the iron-doped molybdenum disulfide nanomaterial modified with DNA according to claim 2, characterized in that: The steps include: The iron-doped molybdenum disulfide nanomaterial according to claim 1 is incubated with DNA in a fixed buffer.

5. The use of the iron-doped molybdenum disulfide nanomaterial according to claim 1 or the iron-doped molybdenum disulfide nanomaterial modified with DNA according to claim 2, comprising any one or any combination of the following: 1) preparing peroxidase-like enzymes or peroxidase mimetic enzymes; 2) preparing materials, sensing units or sensors for protein detection; 3) Preparation of medical, sanitary materials or auxiliary materials; 4) Materials or auxiliary materials for preparing teeth and periodontal tissues; 5) Prepare filling materials or auxiliary filling materials for teeth and periodontal tissues.

6. A product containing the iron-doped molybdenum disulfide nanomaterial according to claim 1 or the iron-doped molybdenum disulfide nanomaterial modified with DNA according to claim 2, comprising any one or any combination of the following: 1) Peroxidase-like enzymes or peroxidase-mimicking enzymes; 2) Materials or sensing units or sensors for protein detection; 3) Medical, sanitary materials or auxiliary materials; 4) Materials or auxiliary materials for teeth and periodontal tissues; 5) Filling materials or auxiliary filling materials for teeth and periodontal tissues.

7. A method for detecting a protein, characterized in that: The steps include: The sample to be tested, the iron-doped molybdenum disulfide nanomaterial according to claim 1 and DNA are incubated in a fixed buffer, or the sample to be tested and the iron-doped molybdenum disulfide nanomaterial modified with DNA according to claim 2 are incubated in a fixed buffer, and then H2O2 and 3,3',5,5'-tetramethylbenzidine are added for incubation, and the absorbance value at 652nm is measured to perform qualitative and / or quantitative detection of the protein.

8. A method for detecting protein by a sensor array, characterized in that the steps include: (1) placing the sample to be tested, the iron-doped molybdenum disulfide nanomaterial described in claim 1 and DNA in an incubation well with a fixed buffer added to a sensor unit constituting a sensor array for incubation, or placing the sample to be tested and the iron-doped molybdenum disulfide nanomaterial modified with DNA described in claim 2 in an incubation well with a fixed buffer added to a sensor unit constituting a sensor array for incubation, then adding H2O2 and 3,3',5,5'-tetramethylbenzidine for incubation, and measuring the absorbance value at 652 nm; (2) Data analysis is performed on the absorbance value at 652 nm to perform qualitative and / or quantitative detection of single target proteins and mixed proteins.

9. The method according to claim 8, characterized in that In step (1), the sensing units are different and the modified DNA is different.

10. The method according to claim 8 or 9, characterized in that: The single target protein comprises bovine hemoglobin, concanavalin A, cytochrome C, human serum albumin, chymosin A, pepsin, papain, and proteinase K, and the mixed protein is a binary mixture of cytochrome C and chymosin A.

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