CN114166909A - MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof - Google Patents
MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof Download PDFInfo
- Publication number
- CN114166909A CN114166909A CN202111315670.9A CN202111315670A CN114166909A CN 114166909 A CN114166909 A CN 114166909A CN 202111315670 A CN202111315670 A CN 202111315670A CN 114166909 A CN114166909 A CN 114166909A
- Authority
- CN
- China
- Prior art keywords
- mos
- nano composite
- zno nano
- electrochemical impedance
- composite antibacterial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 59
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 43
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims abstract description 42
- 229910052961 molybdenite Inorganic materials 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000000243 solution Substances 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 239000008367 deionised water Substances 0.000 claims abstract description 22
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 22
- 230000004048 modification Effects 0.000 claims abstract description 18
- 238000012986 modification Methods 0.000 claims abstract description 18
- 229920000557 Nafion® Polymers 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 10
- 238000012546 transfer Methods 0.000 claims abstract description 9
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 230000006870 function Effects 0.000 claims abstract description 6
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 6
- 241000192125 Firmicutes Species 0.000 claims abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 claims description 28
- 239000000463 material Substances 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000002135 nanosheet Substances 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 12
- -1 polytetrafluoroethylene Polymers 0.000 claims description 11
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 11
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 10
- 239000004246 zinc acetate Substances 0.000 claims description 10
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000011049 filling Methods 0.000 claims description 7
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 239000011733 molybdenum Substances 0.000 claims description 6
- 229910052750 molybdenum Inorganic materials 0.000 claims description 6
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 claims description 6
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea group Chemical group NC(=S)N UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 6
- 239000011701 zinc Substances 0.000 claims description 6
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000002105 nanoparticle Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000004201 L-cysteine Substances 0.000 claims description 4
- 235000013878 L-cysteine Nutrition 0.000 claims description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 239000011593 sulfur Substances 0.000 claims description 4
- 238000004448 titration Methods 0.000 claims description 4
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 3
- 239000011609 ammonium molybdate Substances 0.000 claims description 3
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 3
- 229940010552 ammonium molybdate Drugs 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 239000011684 sodium molybdate Substances 0.000 claims description 3
- 235000015393 sodium molybdate Nutrition 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- 239000003792 electrolyte Substances 0.000 claims description 2
- 239000006210 lotion Substances 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims 5
- 230000003115 biocidal effect Effects 0.000 claims 1
- 238000010276 construction Methods 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 3
- 230000009977 dual effect Effects 0.000 abstract description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N zinc oxide Inorganic materials [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 74
- 239000011787 zinc oxide Substances 0.000 description 49
- 241000191967 Staphylococcus aureus Species 0.000 description 9
- 238000011534 incubation Methods 0.000 description 8
- 239000000725 suspension Substances 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 description 5
- 239000013078 crystal Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000000634 powder X-ray diffraction Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 2
- 108010013639 Peptidoglycan Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004098 selected area electron diffraction Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000446313 Lamella Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000001453 impedance spectrum Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- ABGZKEOYRQFMCR-OXIHULNRSA-M sodium;(2r,3r,4r,5r)-hexane-1,2,3,4,5,6-hexol;chloride Chemical compound [Na+].[Cl-].OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO ABGZKEOYRQFMCR-OXIHULNRSA-M 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- DJWUNCQRNNEAKC-UHFFFAOYSA-L zinc acetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O DJWUNCQRNNEAKC-UHFFFAOYSA-L 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The invention discloses a MoS2Preparation method of-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and MoS prepared by method2Injecting deionized water and nafion solution accounting for 6% of the mixed solution by mass into a tube, and performing ultrasonic treatment to uniformly disperse the deionized water and the nafion solution to obtain electrode modification liquid; transferring 6 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nanocomposite antibacterial-electrochemical impedance non-enzymatic bacterial sensor; MoS of the invention2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and the preparation method and application thereof have the dual functions of resisting bacteria and detecting bacteria for gram-positive bacteria, and have the advantages of low cost, low construction difficulty, simple operation and easy storage.
Description
Technical Field
The invention belongs to the technical field of functional nano materials and electrochemistry, and particularly relates to a MoS2-ZnO nanocomposite germicidalMaterials, electrochemical impedance non-enzymatic bacterial sensors, and methods of making and using the same.
Background
In the present society, harm caused by pathogenic bacteria is proved for countless times, bacterial infection is the most common part of clinical infectious diseases in the present medicine, and the high-efficiency and simple detection of bacteria is the first step for controlling bacteria.
Since 1676 years of microscopic observation of a single microorganism, the monitoring of bacteria has become a persistent subject faced by scientists, but the existing bacteria detection means such as plate method, enzyme-linked immunosorbent assay and the like either need a long-time enrichment and purification process, or need expensive instruments and professional operations, or only can detect bacteria which are thoroughly researched, have high detection threshold and high cost, and are difficult to popularize in wider use scenes, and meanwhile, the detection technology is generally single in function, and needs a quicker, more accurate and more convenient detection method in actual detection.
In the field of biochemistry, electrochemical analysis has the advantages of high sensitivity, fast response, good selectivity, convenient operation, low instrument cost and the like, and attracts the common attention of researchers. Therefore, the development of a simple and sensitive sensor for detecting bacteria is a great trend in the field of bacteria detection.
However, most of the common bacterial electrochemical sensors at present need the participation of bioactive substances such as antibodies, enzymes and the like, the experimental process is complex, the limitation of the existing biotechnology is limited, the cost for purifying and separating the bioactive substances is high, and the test cost is greatly increased.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a MoS2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and the preparation method and application thereof have the dual functions of resisting bacteria and detecting bacteria for gram-positive bacteria, and have the advantages of low cost, low construction difficulty, simple operation and easy storage.
In order to achieve the purpose, the invention adopts the following technical scheme:
MoS2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor comprises the following steps:
step one, mixing a molybdenum source and a sulfur source according to the mass ratio of 3:7, dissolving the mixture in deionized water, performing ultrasonic dispersion, transferring the mixture to a polytetrafluoroethylene lining, filling the polytetrafluoroethylene lining into a hydrothermal kettle, putting the hydrothermal kettle into an oven, performing hydrothermal reaction at the temperature of 200-240 ℃ for 20-24 hours, naturally cooling after the reaction is finished, centrifuging, washing and drying to prepare the MoS2Nanosheets;
step two, weighing zinc acetate and MoS accounting for 2-3% of zinc acetate by mass2Dispersing the nanosheets in ethanol, uniformly stirring, condensing and refluxing for 1-3 h at 60-100 ℃, and preparing the zinc acetate-MoS with the mass concentration of 10-20%2A nanosheet solution;
dropwise adding 0.03g/L LiOH-ethanol solution while stirring, continuously stirring for 20-50 min after titration, adding deionized water, stirring, standing for 5-20 min, pouring out supernatant, washing with a lotion, centrifuging, and drying to obtain MoS2-ZnO nanocomposite bactericidal material;
wherein, the addition amount of LiOH-ethanol solution and zinc acetate-MoS2The volume ratio of the nanosheet mixed solution to the deionized water is 1:1: 1.5;
step three, MoS is weighed2Adding deionized water into a round-bottom centrifuge tube made of the ZnO nano composite antibacterial material, injecting the deionized water into a nafion solution injection tube accounting for 4-8% of the volume fraction of the mixed solution, and performing ultrasonic treatment to uniformly disperse the solution to obtain an electrode modification solution; wherein MoS2The concentration of the-ZnO nano composite antibacterial material is 3-6 g/L;
step four, transferring 4-8 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor.
Preferably, the molybdenum source is one or more of ammonium molybdate, sodium molybdate, molybdenum trioxide or molybdenum hexacarbonyl;
the sulfur source is thiourea or L-cysteine.
Preferably, the filling ratio of the polytetrafluoroethylene lining in the step one is 40-60%.
Preferably, the ultrasonic dispersion is to place the mixed solution in an ultrasonic cleaner for 5-10 min by ultrasonic treatment.
Preferably, the washing and centrifuging are performed repeatedly and repeatedly at 6000-8000 rpm for 5 times by using alcohol water.
Preferably, the drying is to place the sample in an oven at 50 ℃.
The invention also provides the MoS prepared by the method2-ZnO nanocomposite antibacterial-electrochemical impedance non-enzymatic bacterial sensor, said MoS2The ZnO nano composite material monomers are connected and alternately grown to form an intricate and porous island-shaped network, and ZnO nano particles uniformly grow on the sheet-shaped stacked MoS2The above.
Preferably, the antibacterial and bacteria detecting double functions are achieved.
The invention also protects the MoS2Application of-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor in gram-positive bacteria detection.
Preferably, MoS is2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor adopts an electrochemical impedance method to detect the bacteria in 5.0mmol/L (1:1) K in PBS as a solvent3[Fe(CN)6]-K4[Fe(CN)6]And EIS signal response effect in 0.1mol/L KCl electrolyte.
Compared with the prior art, the invention has the following technical effects:
MoS in the invention2MoS of-ZnO nanocomposites2The antibacterial agent has the characteristics of large specific surface area, a large number of active sites, high stability, biocompatibility, broad-spectrum and efficient antibacterial activity, difficulty in generating drug-resistant strains and the like; the nano zinc oxide is one of the most studied inorganic nano antibacterial agents, and the sterilization mechanism mainly comprises that Zn ions are released to destroy the structure and the physiological activity of thalli, the Zn ions interact with the surface of bacteria to destroy cell walls, and active oxygen ROS with the sterilization effect is induced and generated in cells; meanwhile, the nano zinc oxide has good biocompatibilitySex and higher biological activity, immunoregulation ability and high absorption rate; the gram-positive bacteria have a thick peptidoglycan multilayer network macromolecular structure, have certain viscosity and are easy to be firmly combined with a nano plane, and the MoS used by the invention2The ZnO nano composite material has a relatively large and uniform surface, is easy to combine with a peptidoglycan structure, achieves the purpose of detecting bacteria through the interaction of the nano material and the bacteria and the linear relation between an electrochemical impedance signal and the concentration of the bacteria, generates active oxygen through the molybdenum disulfide and the nano zinc oxide together to play an antibacterial role, and has double functions of efficiently sterilizing and detecting the bacteria;
the zinc oxide-loaded molybdenum disulfide antibacterial nano composite material has a certain detection effect on bacterial liquids with different concentrations under the conditions that the incubation time is more than 30min and the pH is 7.0-7.5;
MoS prepared by the invention2The bacterial electrochemical sensor constructed by the ZnO nano composite material does not need bioactive substances such as enzyme, antibody and the like, and has the advantages of low cost, low construction difficulty, simple operation and easy storage;
the invention provides certain application value and reference value for further exploring the field of bacteria detection of nano antibacterial materials in the electrochemical biosensor as basic research.
Drawings
FIG. 1 shows ZnO nanoparticles and different MoS2Addition amount of MoS2-X-ray powder diffraction pattern of ZnO nanocomposite;
FIG. 2 shows MoS2X-ray powder diffraction pattern of (a);
FIG. 3 is the synthesis of MoS in example 12The TEM image of the 100nm transmission electron microscope;
FIG. 4 shows the synthesis of MoS in example 12The 10nm transmission electron microscope TEM picture;
FIG. 5 is a diffraction lattice diagram of the selected area of FIG. 4;
FIG. 6 shows MoS in example 12-Scanning Electron Microscope (SEM) photographs of ZnO nanocomposites;
FIG. 7 is a blank control group of Staphylococcus aureus;
FIG. 8 is a photograph showing the growth of Staphylococcus aureus treated with ZnO nanoparticles;
FIG. 9 shows MoS in example 12A picture of the growth condition of staphylococcus aureus after the nanosheet treatment;
FIG. 10 shows the MoS obtained in example 22-a picture of the growth of staphylococcus aureus after being treated by the ZnO nano composite antibacterial material;
FIG. 11 shows the MoS obtained in example 32-a picture of the growth of staphylococcus aureus after being treated by the ZnO nano composite antibacterial material;
FIG. 12 shows the MoS obtained in example 12-a picture of the growth of staphylococcus aureus after being treated by the ZnO nano composite antibacterial material;
FIG. 13 is an impedance profile of the modified electrode after incubation with the same concentration of bacteria for different periods of time;
FIGS. 14 and 15 are impedance maps of bacteria solutions with different concentrations and incubation time of 30 min;
FIG. 16 shows MoS2Schematic diagram of preparation process of-ZnO nano composite antibacterial-electrochemical impedance non-enzyme bacteria sensor.
Detailed Description
The present invention will be explained in further detail with reference to examples.
Example 1
Firstly, mixing ammonium molybdate and L-cysteine according to the mass ratio of 3:7, dissolving the mixture in 50ml of deionized water, placing the mixture in an ultrasonic cleaner for ultrasonic treatment for 5min, transferring the mixture to a polytetrafluoroethylene lining, placing the lining into a hydrothermal kettle with the filling ratio of 50%, placing the kettle into an oven, carrying out hydrothermal reaction for 22h at 220 ℃, naturally cooling after the reaction is finished, repeatedly centrifuging and washing the mixture for 5 times at 6000rpm alternately by using alcohol water, placing the mixture in the oven at 50 ℃ for drying, and preparing MoS2Nanosheets;
step two, weighing zinc acetate and MoS prepared in step one and accounting for 3% of zinc acetate by mass2Dispersing 1g of nano-sheets in 100ml of ethanol, uniformly stirring, condensing and refluxing at 80 ℃ for 2h, dropwise adding 100ml of LiOH-ethanol solution while stirring, and continuously stirring for 30min after the titrationAdding 150ml of deionized water, stirring, standing for 10min, pouring out supernatant, repeatedly centrifuging at 6000rpm with alcohol-water alternately, washing for 5 times, and oven drying at 50 deg.C to obtain MoS2-ZnO nanocomposite bactericidal material;
step three, weighing 4mg MoS2Putting the ZnO nano composite antibacterial material in a 1.5mL round-bottom centrifuge tube, injecting 1mL deionized water and 60 mu L nafion solution into the centrifuge tube, and putting the centrifuge tube in an ultrasonic cleaner for 5min to uniformly disperse the deionized water and the nafion solution to obtain an electrode modification solution; wherein MoS2The concentration of the-ZnO nano composite antibacterial material is 4 g/L;
step four, transferring 6 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor.
Example 2
Step one, mixing and dissolving sodium molybdate and thiourea according to the mass ratio of 3:7 in 50ml of deionized water, placing the mixture in an ultrasonic cleaner for ultrasonic treatment for 8min, transferring the mixture to a polytetrafluoroethylene lining, placing the polytetrafluoroethylene lining into a hydrothermal kettle with the filling ratio of 40%, placing the polytetrafluoroethylene lining into an oven, carrying out hydrothermal reaction for 24h at 200 ℃, naturally cooling after the reaction is finished, repeatedly centrifuging and washing the mixture for 5 times at 7000rpm alternately by using alcohol water, placing the mixture in the oven at 50 ℃ for drying, and preparing the MoS2Nanosheets;
step two, weighing zinc acetate and MoS prepared in step one and accounting for 2% of zinc acetate by mass2Dispersing 1.5g of nano sheets in 100ml of ethanol, uniformly stirring, condensing and refluxing at 60 ℃ for 3 hours, dropwise adding 100ml of LiOH-ethanol solution while stirring, continuously stirring for 20min after titration, adding 150ml of deionized water, stirring, standing for 5min, pouring out supernatant, repeatedly centrifuging and washing 5 times at 7000rpm by using alcohol water alternately, drying in a 50 ℃ oven to obtain MoS2-ZnO nanocomposite bactericidal material;
step three, weighing 3mg MoS2Putting the ZnO nano composite antibacterial material in a 1.5mL round-bottom centrifuge tube, then taking deionized water and 40 mu L nafion solution to inject into the tube, and putting the tube in an ultrasonic cleaner for ultrasonic treatment for 8min to uniformly disperse the solution to obtain an electrode modification solution; wherein MoS2The concentration of the-ZnO nano composite antibacterial material is 3 g/L;
step four, transferring 4 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor.
Example 3
Step one, mixing molybdenum trioxide and L-cysteine according to the mass ratio of 3:7, dissolving the mixture in 50ml of deionized water, placing the mixture in an ultrasonic cleaning instrument for ultrasonic treatment for 10min, transferring the mixture into a polytetrafluoroethylene lining, placing the lining into a hydrothermal kettle, placing the kettle into an oven with the filling ratio of 60%, performing hydrothermal reaction for 20h at 240 ℃, naturally cooling after the reaction is finished, repeatedly centrifuging and washing the mixture for 5 times at 8000rpm by using alcohol water alternately, placing the mixture in the oven at 50 ℃ for drying, and preparing the MoS2Nanosheets;
step two, weighing zinc acetate and MoS prepared in step one and accounting for 2.5 mass percent of zinc acetate2Dispersing 2g of nano sheets in 100ml of ethanol, uniformly stirring, condensing and refluxing at 100 ℃ for 1h, dropwise adding 100ml of LiOH-ethanol solution while stirring, continuously stirring for 50min after titration, adding 150ml of deionized water, stirring, standing for 20min, pouring out supernatant, repeatedly centrifuging and washing 5 times by using alcohol water alternately at 8000rpm, drying in a 50 ℃ oven to obtain MoS2-ZnO nanocomposite bactericidal material;
step three, weighing 6mg MoS2Putting the ZnO nano composite antibacterial material in a 1.5mL round-bottom centrifuge tube, then taking deionized water and 80 mu L of nafion solution, injecting into the tube, and putting into an ultrasonic cleaner for ultrasonic treatment for 10min to uniformly disperse the deionized water and the nafion solution to obtain an electrode modification solution; wherein MoS2The concentration of the-ZnO nano composite antibacterial material is 6 g/L;
step four, transferring 8 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor.
FIG. 1 shows different MoS2Addition amount of MoS2-X-ray powder diffraction (XRD) pattern of ZnO nanocomposite. The XRD spectrum result shows that the peak position of the XRD spectrum of the composite material is in accordance with the ZnO standardCard correspondence with MoS2The ZnO peak intensity gradually decreases with increasing introduction amount, and FIG. 2 shows MoS2XRD pattern, MoS of nanosheet2The (002), (2 θ ═ 14 °) diffraction peaks of the hexagonal crystal were shifted, the diffraction peaks at 2 θ ═ 32 °, 34 °, and 57 ° corresponded to (100) (102) (110) (2H phase), respectively, and MoS was observed2MoS is known because the diffraction peaks of the nanosheets are weak as a whole2The crystallinity is poor, the XRD peak is covered by the ZnO peak, and the XRD spectrum is not obvious. Thus, MoS2Need to be verified by other means such as SEM.
FIGS. 3 to 6 show the MoS synthesized in this experiment2The Transmission Electron Microscope (TEM) photo shows that the molybdenum disulfide nanosheet prepared by the experiment has thinner lamella and fewer edge layers, is formed by assembling and stacking a plurality of molybdenum disulfide monolayers, and has the interlayer spacing of about 0.63nm corresponding to the hexagonal MoS2The (002) crystal face of the crystal has clear crystal lattice stripes. TEM results were processed with Digital Micrograph software to obtain Selected Area Electron Diffraction (SAED) patterns, which can be seen in FIG. 5, MoS2The crystallinity of the nanosheets ranges from polycrystalline to amorphous, and is poor.
For the prepared MoS2The antibacterial performance of the-ZnO nanocomposite is studied, and a sample is dispersed in PBS buffer solution to 4mg/mL and irradiated under an ultraviolet lamp. 1mL of the suspension was diluted with sterilized PBS buffer until the absorbance (OD) was 0.03. And (3) putting 1mL of diluted bacterial suspension into a 5mL centrifuge tube, adding 1mL of sample into the centrifuge tube, and placing the centrifuge tube into a constant-temperature water bath oscillator at 37 ℃ and the rotating speed of 150r/min to oscillate for 16-20 h to obtain the flat antibacterial experimental bacterial suspension. Pouring the sterilized D-mannitol sodium chloride agar culture medium into a sterilized culture dish, wherein the volume of the culture dish is about one half of that of the culture dish, naturally cooling the culture dish in a super clean bench under ultraviolet irradiation, sucking 50 mu L of the shaken bacterial suspension, inoculating the bacterial suspension onto the solidified culture medium, and respectively and uniformly coating the bacterial suspension by using a sterile coating rod. Placing the coated culture dish and the blank control culture dish without the sample into a constant temperature and humidity box at 37 ℃ for 48-72 h, taking out the culture dishes, taking a colony photo of each culture dish, and as shown in figure 12, showing MoS2When the mass fraction is 3 percent,compared with ZnO nanoparticles shown in figure 8 and MoS2 nanosheet shown in figure 9, the antibacterial effect of the composite material is obviously improved;
MoS prepared in example 12the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor adopts an alternating current impedance method to detect an impedance spectrogram of the sensor after the sensor is incubated in bacterial liquids with different concentrations for different times. FIG. 13 is an impedance spectrum of the modified electrode in the same concentration of bacterial solution before and after different incubation times. The staphylococcus aureus is adsorbed on the surface of the sensor, and the electronic shielding effect of the cell membrane can cause the interface to change, thereby generating the change of an impedance map, RctIt is interface electron transfer resistance, and the electron shielding effect of the biological membrane becomes stronger along with the capture of bacteria, so RctObvious change can be generated, and the change Delta R of the interface charge transfer resistance is obtained by comparisonctThereby realizing the detection of the staphylococcus aureus.
The half-circle evident in the Nyquist plot (Nyquist) shown in fig. 13 corresponds to the reaction resistance of the working electrode, as can be seen from the plot, with MoS2The incubation time of the ZnO nano composite antibacterial material modified electrode in a bacterial solution with the absorbance of about 0.06 is increased, and the arc of the high-intermediate frequency region represents that the charge transfer impedance of the electrode related to the Faraday reaction is also increased in a generally linear manner, because more and more Staphylococcus aureus is captured to the surface of the sensor along with the increase of time, the cell membrane of bacteria has an electronic shielding effect and obstructs the transfer of a redox couple, so that the interface conduction resistance is increased, namely, the EIS can be timely and effectively changed correspondingly along with the increase of the concentration of bacteria adsorbed to the electrode, so that the electronic conduction speed of the surface of the sensor and an electroactive substance is reflected. Thus, R in this experimentctThe size of (d) is correlated with the concentration of the bacteria. The impedance data were further fitted with ZSimDemo software, and a fitting circuit Rs (crq (rw)) was used to observe the specific relationship between the impedance change and the concentration, where the square points in the impedance plot represent the raw data, and the circular points correspond to the fitted data, respectively. As can be seen from the good overlapping relationship, the fitted circuit is more consistent with the experimental result. Estimating the dynamics of an electrode process by means of an analog circuitThe learning mechanism is consistent with the guess. Preferably, when the incubation time is more than 30min, the impedance signal is linear;
FIGS. 14 and 15 are impedance profiles of bacteria solutions of different concentrations with incubation time of 30 min. It can be seen that when the culture medium is incubated for 30min, the linear correlation degree of the impedance signal and the bacterial liquid concentration is good, and the impedance signal and the fitting circuit also show a good overlapping relation. Illustrating the MoS prepared by the invention2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor shows a certain detection effect on bacterial liquids with different concentrations under the conditions that the incubation time is 30min and the pH value is 7.3-7.4, and provides a certain reference value for further exploring a nano antibacterial material in the field of detecting bacteria by an electrochemical biosensor as a basic research.
Claims (10)
1. MoS2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized by comprising the following steps of:
step one, mixing a molybdenum source and a sulfur source according to the mass ratio of 3:7, dissolving the mixture in deionized water, performing ultrasonic dispersion, transferring the mixture to a polytetrafluoroethylene lining, filling the polytetrafluoroethylene lining into a hydrothermal kettle, putting the hydrothermal kettle into an oven, performing hydrothermal reaction at the temperature of 200-240 ℃ for 20-24 hours, naturally cooling after the reaction is finished, centrifuging, washing and drying to prepare the MoS2Nanosheets;
step two, weighing zinc acetate and MoS accounting for 2-3% of zinc acetate by mass2Dispersing the nanosheets in ethanol, uniformly stirring, condensing and refluxing for 1-3 h at 60-100 ℃, and preparing the zinc acetate-MoS with the mass concentration of 10-20%2A nanosheet solution;
dropwise adding 0.03g/L LiOH-ethanol solution while stirring, continuously stirring for 20-50 min after titration, adding deionized water, stirring, standing for 5-20 min, pouring out supernatant, washing with a lotion, centrifuging, and drying to obtain MoS2-ZnO nanocomposite bactericidal material;
wherein, the addition amount of LiOH-ethanol solution and zinc acetate-MoS2The volume ratio of the nanosheet mixed solution to the added deionized water is 1:1: 1.5;
step threeWeighing MoS2Putting the ZnO nano composite antibacterial material into a round-bottom centrifugal tube, then injecting deionized water, finally injecting nafion solution accounting for 4-8% of the volume fraction of the mixed solution, and performing ultrasonic treatment to uniformly disperse the nafion solution to obtain an electrode modification solution; wherein MoS2The concentration of the-ZnO nano composite antibacterial material is 3-6 g/L;
step four, transferring 4-8 mu L of electrode modification liquid by using a liquid transfer gun, and dripping the electrode modification liquid on the surface of the pretreated glassy carbon electrode to obtain MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor.
2. The MoS of claim 1, wherein the MoS is a solid-state imaging device2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that the molybdenum source is one or more of ammonium molybdate, sodium molybdate, molybdenum trioxide or molybdenum hexacarbonyl;
the sulfur source is thiourea or L-cysteine.
3. The MoS of claim 1, wherein the MoS is a solid-state imaging device2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that the filling ratio of the polytetrafluoroethylene lining in the step one is 40-60%.
4. The MoS of claim 1, wherein the MoS is a solid-state imaging device2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that the ultrasonic dispersion is that the mixed solution is placed in an ultrasonic cleaner for ultrasonic treatment for 5-10 min.
5. The MoS of claim 1, wherein the MoS is a solid-state imaging device2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that the washing and centrifuging are repeated and washed for 5 times by using alcohol and water alternately at 6000-8000 rpm.
6. The MoS of claim 1, wherein the MoS is a solid-state imaging device2The preparation method of the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that the baking is carried outDry is to place the sample in a 50 ℃ oven to dry.
7. MoS prepared by the method of any one of claims 1-62-ZnO nanocomposite antibacterial-electrochemical impedance non-enzymatic bacterial sensor, characterized in that said MoS2The ZnO nano composite material monomers are connected and alternately grown to form an intricate and porous island-shaped network, and ZnO nano particles uniformly grow on the sheet-shaped stacked MoS2The above.
8. The MoS of claim 7, wherein2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized by having double functions of antibiosis and bacterial detection.
9. The MoS of claim 72the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is applied to gram-positive bacteria detection.
10. The MoS of claim 92Application of-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor is characterized in that MoS is used2the-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor adopts an electrochemical impedance method to detect the bacteria in 5.0mmol/L (1:1) K in PBS as a solvent3[Fe(CN)6]-K4[Fe(CN)6]And EIS signal response effect in 0.1mol/L KCl electrolyte.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111315670.9A CN114166909B (en) | 2021-11-08 | 2021-11-08 | MoS (MoS)2ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111315670.9A CN114166909B (en) | 2021-11-08 | 2021-11-08 | MoS (MoS)2ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114166909A true CN114166909A (en) | 2022-03-11 |
CN114166909B CN114166909B (en) | 2024-06-21 |
Family
ID=80478588
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111315670.9A Active CN114166909B (en) | 2021-11-08 | 2021-11-08 | MoS (MoS)2ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114166909B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103558271A (en) * | 2013-10-17 | 2014-02-05 | 宁波大学 | Electrochemical biosensor for detecting penicillin and preparation method and application thereof |
CN110618178A (en) * | 2019-09-16 | 2019-12-27 | 天津理工大学 | Preparation method of electrochemical impedance electrode for detecting bacteria |
CN111838148A (en) * | 2020-07-14 | 2020-10-30 | 陕西科技大学 | Preparation method of cobalt-doped zinc oxide/molybdenum disulfide nano composite antibacterial agent |
US20210332489A1 (en) * | 2020-04-27 | 2021-10-28 | Iowa State University Research Foundation, Inc. | Laser-induced graphene electrodes adaptable for electrochemical sensing and catalysis |
-
2021
- 2021-11-08 CN CN202111315670.9A patent/CN114166909B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103558271A (en) * | 2013-10-17 | 2014-02-05 | 宁波大学 | Electrochemical biosensor for detecting penicillin and preparation method and application thereof |
CN110618178A (en) * | 2019-09-16 | 2019-12-27 | 天津理工大学 | Preparation method of electrochemical impedance electrode for detecting bacteria |
US20210332489A1 (en) * | 2020-04-27 | 2021-10-28 | Iowa State University Research Foundation, Inc. | Laser-induced graphene electrodes adaptable for electrochemical sensing and catalysis |
CN111838148A (en) * | 2020-07-14 | 2020-10-30 | 陕西科技大学 | Preparation method of cobalt-doped zinc oxide/molybdenum disulfide nano composite antibacterial agent |
Non-Patent Citations (1)
Title |
---|
王春皓;崔传金;谷学静;陈宏硕;张学超;李静;: "检测金黄色葡萄球菌的微叉指电极阻抗生物传感器研究", 食品研究与开发, no. 10 * |
Also Published As
Publication number | Publication date |
---|---|
CN114166909B (en) | 2024-06-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Parthasarathy et al. | Biocompatible TiO2-CeO2 nano-composite synthesis, characterization and analysis on electrochemical performance for uric acid determination | |
CN108524941A (en) | A kind of enzyme driving ampuliform nano-motor and preparation method thereof | |
JP2007523096A (en) | Metal complex solution and its application | |
Johnson et al. | Urease-mediated room-temperature synthesis of nanocrystalline titanium dioxide | |
Lee et al. | Eco-toxicity of commercial silver nanopowders to bacterial and yeast strains | |
CN106423275B (en) | A kind of magnetic composite for antibiotic in Visible Light Induced Photocatalytic water | |
CN103255177A (en) | Method for preparing graphene simultaneously doped with nitrogen and sulfur through biological reduction | |
CN111088166A (en) | Method for promoting microorganism to induce calcium carbonate precipitation by phycomycete co-culture system | |
CN108546331A (en) | The preparation method of enzyme-metal organic frame composite membrane and the application of modified electrode bio-sensing | |
CN111796013B (en) | Zeolite imidazolate framework/metal oxide/molybdenum sulfide composite material | |
CN105724372B (en) | A kind of support type ZnO antimicrobial composite materials and preparation method thereof | |
CN105738437B (en) | A kind of preparation method and application of the electrochemistry parathion sensor based on metal and metal oxide co-doped nano composite | |
CN105572108A (en) | Preparing method and application of electrochemiluminescence demeton sensor | |
CN116698937A (en) | MnMoO 4 /g-C 3 N 4 Nanocomposite modified electrode, preparation method thereof, electrochemical sensor and application | |
CN106226372A (en) | A kind of modification ITO electrode that can improve product electricity bacterium efficiency of fuel cell generation and preparation method thereof | |
Yang et al. | Intercalation of methylene blue into layered manganese oxide and application of the resulting material in a reagentless hydrogen peroxide biosensor | |
CN101813660B (en) | Method for preparing fixed enzyme electrode by using TiO2 as carrier | |
Satvekar et al. | Multienzymatic cholesterol nanobiosensor using core–shell nanoparticles incorporated silica nanocomposite | |
CN104450564A (en) | Sulfate reducing bacterium capable of being used for preparing Ag/AgCl nano particles | |
CN114166909A (en) | MoS2-ZnO nano composite antibacterial-electrochemical impedance non-enzymatic bacterial sensor and preparation method and application thereof | |
CN105738353B (en) | A kind of preparation method and application of optical electro-chemistry decis sensor | |
CN107328834A (en) | A kind of composite modified electrode of lead ion detection being used in livestock and poultry drinking water and preparation method thereof | |
CN107519842A (en) | Modified oxidized single angle and its preparation method and application and the method for adsorbing antibiotic | |
CN101051034A (en) | Electrochemical biological sensor sensing film and its preparing method and use | |
CN105486720A (en) | H2S gas sensing material and preparation method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |