CN106854619B - Crosslinking device based on plasma, using method and application - Google Patents
Crosslinking device based on plasma, using method and application Download PDFInfo
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- CN106854619B CN106854619B CN201710043758.7A CN201710043758A CN106854619B CN 106854619 B CN106854619 B CN 106854619B CN 201710043758 A CN201710043758 A CN 201710043758A CN 106854619 B CN106854619 B CN 106854619B
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
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- Hematology (AREA)
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- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The application relates to a plasma-based crosslinking device, a using method and an application thereof, wherein the plasma-based crosslinking device comprises a reaction cavity, a plasma generating device and a sample placing area to be treated are arranged in the reaction cavity, and the plasma generating device generates plasma to enable covalent crosslinking of proteins and proteins, nucleic acids and/or proteins and nucleic acids in the sample to be treated. The crosslinking device provided by the application can be used in various fields such as biochemical engineering, biomedicine and the like.
Description
Technical Field
The application relates to a plasma-based crosslinking device, a method for using the device and the application thereof in the field of biotechnology.
Background
Modern life science has entered the era of molecular biology, and research on life phenomena has reached the molecular level. Many experiments have shown that many cellular vital activities involve interactions between specific DNA segments and specific protein factors, such as DNA replication and repair, RNA transcription and modification, viral transfection and proliferation, etc. Therefore, it is of increasing importance to study the interaction of proteins and DNA by specific covalent cross-linking of the DNA and proteins.
Existing methods for studying protein and nucleic acid interactions mainly include ultraviolet crosslinking and formaldehyde crosslinking. The ultraviolet crosslinking instrument is the most mainstream instrument used in the direction at present, and the principle is as follows: in vivo, proteins and nucleic acids form non-covalent bonds, and if such bonds are irradiated by ultraviolet light, covalent crosslinks can be formed between pyrimidine bases in the DNA molecule and various amino acid residues in the protein molecule (e.g., lysine, serine, cysteine, tryptophan, methionine, tyrosine, phenylalanine, arginine, etc.). If the DNA is pre-labeled, the DNA is combined with the DNA binding protein and subjected to ultraviolet irradiation to generate covalent crosslinking, the DNA which is not combined with the protein is removed by DNase, and the rest is the DNA protein covalent binding complex, so that the DNA binding protein and the molecular weight thereof can be detected. However, ultraviolet rays tend to damage the DNA in the sample (e.g., break DNA), affecting the detection results, and the efficiency of uv treatment to produce covalent crosslinks is low. Formaldehyde is highly efficient in producing crosslinks, but is prone to producing many nonspecific crosslinks, and it is difficult to control the reaction time accurately, and to carry out the termination reaction. The conventional crosslinking methods have limitations, and therefore, it is important to develop a device for efficiently and accurately studying interactions between proteins and nucleic acids.
Cryogenic plasmas are partially ionized gases that contain a variety of physical and chemical effects, such as ultraviolet radiation, electromagnetic fields, thermal effects, charged particles, reactive particles, and the like. At present, low-temperature plasmas in the fields of water, air, food packaging materials, food, grain and the like have great potential. For example, CN102068912a discloses a method for preparing a negatively charged nanofiltration membrane by plasma radiation initiated grafting, CN103835002B discloses a method for degumming kenaf by combining low-temperature plasma with biological enzymes, and EP1255616B1 discloses a method for producing a strongly adhesive surface coating by plasma activated grafting. However, the prior art does not disclose the use of plasma technology, in particular atmospheric pressure low temperature plasma technology, in the field of nucleic acid and/or protein cross-linking.
Disclosure of Invention
Summary of The Invention
The inventor of the present application has surprisingly found that the interaction between protein and nucleic acid can be studied by using plasma, and the method has a wide application prospect. The present application utilizes active particle components in plasma to covalently crosslink proteins and nucleic acids to prepare a plasma crosslinking device. The experimental result shows that the plasma crosslinking device has high crosslinking efficiency, can generate obvious covalent crosslinking within two minutes, has relatively small damage to DNA molecules in the combination, has less nonspecific crosslinking, and has the advantages of high efficiency, accuracy, no pollution and the like.
Based on the above, the application discloses a plasma-based crosslinking device, which comprises a reaction cavity, wherein a plasma generating device and a sample placement area to be treated are arranged in the reaction cavity, and the plasma generating device generates plasma to enable covalent crosslinking of proteins and proteins, nucleic acids and/or proteins and nucleic acids in the sample to be treated.
Detailed description of the application
Embodiments of various aspects herein may be described by the following numbered paragraphs.
1. The plasma-based crosslinking device is characterized by comprising a reaction cavity 1, wherein a plasma generating device 2 and a sample placement area 3 to be treated are arranged in the reaction cavity 1, and the plasma generated by the plasma generating device 2 can be used for covalently crosslinking proteins, nucleic acids and/or proteins and nucleic acids in the sample to be treated.
2. The plasma-based crosslinking apparatus of paragraph 1, wherein the reaction chamber 1 further comprises a bottom plate and a top cover, and a front cover, a rear cover, a left side plate and a right side plate perpendicular to the bottom plate, the top cover, the front cover, the rear cover, the left side plate and the right side plate enclosing a closed chamber;
optionally, the bottom plate, the top cover, the front cover, the rear cover, the left side plate and/or the right side plate are provided with an air inlet 4 and/or an air outlet 5.
3. The plasma-based crosslinking apparatus of any one of paragraphs 1 or 2, wherein the plasma generating apparatus 2 comprises a plate-shaped high-voltage electrode 9, an insulating dielectric plate 6 and a mesh-shaped ground electrode 7, wherein the plate-shaped high-voltage electrode 9 is at the uppermost layer, the insulating dielectric plate 6 is in the middle, the mesh-shaped ground electrode 7 is at the bottom layer facing the inside of the cavity of the reaction chamber 1, and the plate-shaped high-voltage electrode 9 is connected to a high-voltage line.
4. The plasma-based crosslinking apparatus of paragraph 3, wherein the plate-like high voltage electrode 9 is capable of adjusting discharge frequency and voltage;
preferably, the voltage range of the plate-shaped high-voltage electrode 9 is between 1kV and 20kV, the frequency range is less than or equal to 100kHz, and the plate-shaped high-voltage electrode is one of pulse, direct current, alternating current and radio frequency power sources.
5. A plasma-based crosslinking apparatus as claimed in any one of the preceding paragraphs, wherein the plasma is a low temperature plasma.
6. The plasma-based crosslinking apparatus of any one of the preceding paragraphs, wherein the nucleic acid is DNA and/or RNA.
7. A plasma-based cross-linking device according to any one of the preceding paragraphs, wherein the protein is any one or more proteins in a biological sample.
8. The plasma-based crosslinking apparatus of any one of the preceding paragraphs, wherein the material of the plate-like high-voltage electrode 9 is a conductive material.
9. The plasma-based crosslinking apparatus of paragraph 8, wherein the plate-like high voltage electrode 9 is made of red copper or stainless steel.
10. The plasma-based crosslinking apparatus of any one of the preceding paragraphs, wherein the material of the insulating dielectric sheet 6 is a high voltage resistant insulating organic or inorganic material.
11. The plasma-based crosslinking apparatus of paragraph 10, wherein the material of the dielectric sheet 6 is polytetrafluoroethylene, ceramic, rubber, glass fiber reinforced plastic, crosslinked polystyrene, plexiglass, epoxy, polyethylene, polycarbonate, quartz glass, mica board, plated board, or polyimide.
12. The plasma-based crosslinking apparatus of any one of the preceding paragraphs, wherein the material of the mesh-like ground electrode 7 is an electrically conductive material.
13. The plasma-based crosslinking apparatus of paragraph 12, wherein the mesh-shaped ground electrode 7 is made of alloy steel or plated steel.
14. The plasma-based crosslinking apparatus as claimed in any one of the preceding paragraphs, wherein the sample-placement area 3 to be treated comprises a lift table 10, and the distance between the sample to be treated and the plasma-generating device 2 is adjusted by varying the height of the lift table 10.
15. The plasma-based crosslinking apparatus of paragraph 14, wherein the height of the lift table 10 is varied such that the distance between the sample to be treated and the plasma generating device 2 is 0.5-2cm; preferably 1cm.
16. The plasma-based crosslinking apparatus of any of the preceding paragraphs, wherein the sample to be treated is a biological sample or a solution for a biological sample.
17. The plasma-based crosslinking apparatus of paragraph 16, wherein the biological sample is one or more of a microbial fluid, a cell culture, an animal or plant tissue, a nucleic acid extract, a protein extract, an in vitro protein and nucleic acid mixture; the solution for biological samples is physiological saline, phosphate buffer, or the like.
18. The plasma-based crosslinking apparatus of any one of the preceding paragraphs, further comprising a gas flow controller module 11 coupled to the reaction chamber 1, the gas flow controller module 11 providing a reactant gas to the plasma-generating device 2.
19. The plasma-based crosslinking apparatus of paragraph 18, wherein the gas flow controller module 11 comprises a controller 12, a gas inlet 13 and a gas outlet 14, wherein the controller 12 adjusts the desired working gas species, gas flow and ratio by computer control, wherein the gas outlet 14 is connected to the plasma crosslinking apparatus.
20. The plasma-based crosslinking apparatus of paragraphs 18 or 19, wherein the reactant gas provided by the gas flow controller module 11 is one or more of air, helium, argon, nitrogen, oxygen, and mixtures thereof.
21. The plasma-based crosslinking apparatus of paragraph 20, wherein the reactant gas provided by the gas flow controller module 11 is helium.
22. A plasma-based crosslinking apparatus as claimed in any one of the preceding paragraphs, wherein: the device also comprises a timing function module and/or a voltage selection function module, wherein the timing function module can set the processing time, and the voltage selection function module can set different voltages to adjust the discharge power.
23. A method of using the crosslinking apparatus of any one of paragraphs 1-22.
24. The method of paragraph 23, comprising the steps of:
the first step: processing the material to obtain a sample to be processed;
and a second step of: placing a sample to be treated in a sample placing area 3 to be treated of the reaction cavity 1 of the crosslinking device;
and a third step of: the plasma generating device 2 generates plasma;
fourth step: the proteins in the sample to be treated are covalently cross-linked with proteins, nucleic acids with nucleic acids, and/or proteins with nucleic acids.
25. The method of paragraph 23, wherein the first step:
the material is a microbial bacterial liquid and a suspension cell culture, the processing steps comprise the steps of collecting the microbial or suspension cell culture, suspending the microbial or suspension cell culture in physiological saline, pouring the microbial or suspension cell culture into a plate to form a thin layer with the thickness of 1-2mm, and obtaining a sample to be processed; or (b)
2, the material is an adherent cell culture, and the treatment step comprises pouring out a culture solution in the adherent cell culture, washing with physiological saline for 1-2 times, and then covering with 1-2mm physiological saline to obtain a sample to be treated; or (b)
3, the material is animal and plant tissue, the treatment step comprises cutting the animal and plant tissue into thin layers with the thickness of 1-2mm, and placing the thin layers into a plate to obtain a sample to be treated; or (b)
4, the material is an extracellular sample, the processing step comprises adding extracellular protein, nucleic acid or a sample obtained by mixing the protein and the nucleic acid into a plate to form a thin layer with the thickness of 1-2mm, and obtaining the sample to be processed.
26. The method of paragraphs 24-25, wherein covalent crosslinking is carried out for a period of 1-5 minutes, preferably 2 minutes.
27. A protein cross-linked product obtained by the method of paragraphs 23-26.
28. A nucleic acid crosslinked product obtained by the method of paragraphs 23-26.
29. A protein nucleic acid crosslinked product obtained by the method of paragraphs 23-26.
30. Use of the crosslinking apparatus of paragraphs 1-22 in the field of biology.
31. The use of paragraph 30, wherein the biological domain is a biochemical domain or a biomedical domain.
Technical terms:
the terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, and the endpoints recited.
When describing a measurable value, such as a parameter, amount, time period, etc., the term "about" as used herein is intended to encompass variations of + -20% or less, preferably + -10% or less, more preferably + -5% or less, more preferably + -1% or less, more preferably + -0.1% or less, from the specified value, such variations being suitable for use in the disclosed application.
The beneficial effects are that:
(1) The application does not need other additives and can not pollute biological samples;
(2) The application has the timing function and the voltage selecting function;
(3) The application adopts a creeping discharge mode, can be contacted with a biological sample to be treated in a large area, and improves the treatment efficiency;
(4) By adjusting the frequency or voltage of the plate-shaped high-voltage electrode, the gas entering speed and the plasma generating speed can be synchronously adjusted, so that the density of the active ingredients is high, and the treatment efficiency is higher;
(5) The application reduces the serious fracture damage to DNA molecules in the crosslinking compound, has higher crosslinking efficiency, and can form covalent crosslinking in two minutes;
(6) The plasma generating device is operated under the atmospheric pressure, a vacuum cavity is not needed, the equipment is simple, the operation is convenient, the cost is low, and toxic residues are not generated after the plasma treatment;
the foregoing is merely illustrative of some aspects of the present application and is not, nor should it be construed as limiting the application in any respect.
All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto without departing from the spirit or scope of the application. The following examples further illustrate the application in detail and are not to be construed as limiting the scope of the application or the particular methods described herein.
Drawings
The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate the application and together with the description serve to explain the preferred embodiment, without limitation to the application, wherein:
fig. 1: a structural diagram of the plasma crosslinking device;
fig. 2: structure diagram of plasma generating device;
fig. 3: a block diagram of a plasma crosslinking apparatus with a lift table and a gas flow controller module;
fig. 4: structure of the gas flow controller module;
fig. 5: agarose gel electrophoresis of protein and DNA mixtures treated in vitro using a plasma crosslinking device.
Description of the reference numerals
1 reaction chamber 2 plasma generating device
3. Sample to be treated places district 4 air inlet
5. Air outlet 6 insulating medium plate
7 netted grounding electrode 8 high-voltage wire
9. Plate-shaped high-voltage electrode 10 lifting platform
11 gas flow controller module 12 controller
13 gas inlet 14 gas outlet
Detailed Description
The advantages and features of the present application will become more apparent from the following description of the embodiments taken in conjunction with fig. 1-5. These examples are merely exemplary and do not limit the scope of the application in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present application may be made without departing from the spirit and scope of the present application, but these changes and substitutions fall within the scope of the present application.
Example 1 plasma crosslinking apparatus
The plasma crosslinking device mainly comprises a reaction cavity 1, wherein the reaction cavity 1 can be cuboid, cubic or other shapes, a plasma generating device 2 and a sample placing area 3 to be processed are arranged in the reaction cavity 1, and the plasma generated by the plasma generating device 2 can enable proteins and proteins, nucleic acids and/or proteins and nucleic acids in the sample to be processed to be covalently crosslinked. The sample to be treated placing area 3 is used for placing the sample to be treated. The reaction chamber 1 includes a bottom plate and a top cover, and a front cover, a rear cover, a left side plate and a right side plate perpendicular to the bottom plate, and the bottom plate, the top cover, the front cover, the rear cover, the left side plate and the right side plate may enclose a closed chamber. The gas inlet 4 and the gas outlet 5 are respectively arranged at one end and the other end of the reaction chamber 1, and the plasma generating device 2 is fixed at the upper part of the reaction chamber 1. In the plasma generating device 2, namely, a plate-shaped high-voltage electrode 9, an insulating medium plate 6 and a net-shaped grounding electrode 7 are laminated together to serve as a top cover of the reaction cavity, the plate-shaped high-voltage electrode 9 is arranged at the uppermost layer, the insulating medium plate 6 is arranged in the middle, the net-shaped grounding electrode 7 faces the inside of the cavity of the reaction cavity 1 at the bottom layer, and the high-voltage electrode is connected with a high-voltage line.
When in operation, a plurality of gases such as air, helium, argon, nitrogen and/or oxygen and the like are added from the air inlet 4, and the gases are mixed at different proportions. The introduced gas is ionized at the ion generating device 2 to generate plasma, active particles in the plasma are diffused into the cavity of the reaction cavity 1 again, and the biological sample in the sample placing area 3 to be processed is processed.
The plasma generating device 2 includes a plate-like high-voltage electrode 9, an insulating dielectric plate 6, and a mesh-like ground electrode 7. Wherein the plate-shaped high voltage electrode 9 is used for adjusting the discharge frequency and voltage. The plate-shaped high-voltage electrode 9 is connected to a high-voltage power supply, and the plasma generator 2 discharges a working gas to generate plasma when excited by the high-voltage power supply. According to experimental requirements, the voltage range of the plate-shaped high-voltage electrode 9 is between 1kV and 20kV, and the frequency range is less than or equal to 100kHz.
The insulating dielectric plate 6 may be made of an insulating dielectric material such as polytetrafluoroethylene, ceramic, rubber, plastic, glass, and/or a plating plate. The mesh-shaped grounding electrode 7 can increase the volume of the plate-shaped high-voltage electrode 9, the insulating medium plate 6 and the mesh-shaped grounding electrode 7, the area and the number of the electrodes in the plasma generating device 2 according to experimental requirements, and the amount of the disposable biological sample treatment can be increased, so that the treatment efficiency can be improved.
The high voltage power supply may also be a pulsed high voltage power supply with a pulse frequency not higher than 100kHz. The higher the frequency of the pulsed high voltage power supply, the faster the plasma processing speed and the lower the discharge voltage, but the plasma temperature will increase with frequency. With the technical scheme, the generation efficiency of active particles is higher under the excitation of a pulse high-voltage power supply, and meanwhile, the plasma temperature is further reduced due to the reduction of the total discharge power.
Example 2 plasma crosslinking apparatus with an internal Lift Table and/or an external gas flow controller Module
The sample placing area 3 to be treated can be provided with a lifting table 10, and the lifting table 10 can adjust the distance between the sample to be treated and the plasma generating device 2, and the distance is generally 1cm.
When in use, a target biological sample (mainly comprising microbial bacteria liquid, cell culture, animal tissue, in-vitro protein and DNA or a mixture thereof and the like) which needs to be crosslinked is added onto a matched dish, then the dish is placed into a lifting table 10 in a cavity, the height is adjusted, and then plasma is generated for treatment.
The plasma crosslinking apparatus may also be externally connected to a gas flow controller module 11, the gas flow controller module 11 serving to provide the reactive gas to the plasma generating apparatus 2. The gas flow controller module 11 comprises a controller 12, a gas inlet 13 and a gas outlet 14, wherein the controller 12 is controlled by a computer, wherein the gas outlet 14 is connected to the gas inlet 4 of the plasma cross-linking device. The gas flow controller module 11 can control the kinds of the working gases for generating the plasma through the computer and the controller 12, and adjust the flow rate and the proportion of each gas.
In addition, the plasma crosslinking device can further comprise a timing function module and a voltage selection function module, wherein the timing function module can set processing time, and the voltage selection function module can set different voltages to adjust the discharge power.
EXAMPLE 3 crosslinking apparatus of plasma crosslinking test of proteins
1. Materials: small molecule proteins (composed of 25 amino acids, synthesized by Ningbo Kangbei Biochemical Co., ltd.).
2. The method comprises the following steps: small molecular protein (0.1 mg/ml), polytetrafluoroethylene is selected as an insulating medium material, and mixed gas of helium and air is used as working gas for discharging to generate low-temperature plasma:
sample 1: treating the mixture sample for 1min respectively to obtain;
sample 2: treating the mixture sample for 2min respectively to obtain the mixture;
50 microliters of sample is added into a 96-well plate, the 96-well plate is placed on a sample placement area to be treated in a cavity, and a power supply is applied with voltage to excite an electrode to discharge so as to generate plasma for treatment.
The molecular weight of the treated sample was detected by MALDI-TOF mass spectrometry.
3. Results
TABLE 1 results of protein crosslinking test by plasma crosslinking apparatus
EXAMPLE 4 crosslinking apparatus of plasma Cross-linking test on protein-DNA
1. Material handling
Sample 3: the method comprises the steps of collecting microorganism bacterial liquid and suspension cell culture, suspending the microorganism or suspension cell culture in normal saline, pouring the suspension cell culture into a plate to form a thin layer with the thickness of 1-2mm, and obtaining a sample to be treated;
sample 4: the method comprises the steps of adhering cell cultures, wherein the treatment step comprises pouring out culture solution in the adhering cell cultures, washing with physiological saline for 1-2 times, and then covering with 1-2mm physiological saline to obtain a sample to be treated;
sample 5: animal and plant tissues are cut into thin layers with the thickness of 1-2mm, and the thin layers are placed in a plate to obtain a sample to be treated;
sample 6: and (3) an extracellular sample, wherein the treatment step comprises the steps of adding extracellular proteins, nucleic acids or a sample obtained by mixing the proteins and the nucleic acids into a plate to form a thin layer with the thickness of 1-2mm, so as to obtain the sample to be treated.
2. Crosslinking reaction using plasma crosslinking apparatus
Polytetrafluoroethylene is used as an insulating medium material, mixed gas of helium and air is used as working gas to discharge to generate low-temperature plasma, the distance between a processed sample and a plasma generating device is 1cm, and the sample is processed for 4 min.
Extracting the genome DNA of the sample 3-5 by adopting a phenol-chloroform extraction method. The treated culture or tissue was collected by centrifugation, suspended in physiological saline, added with an equal volume of phenol-chloroform, thoroughly mixed, centrifuged, and the supernatant was aspirated, and the supernatant was mixed with a loading buffer and subjected to electrophoresis on agarose gel containing 0.5% SDS for electrophoresis analysis. Sample 6 was directly mixed with loading buffer and subjected to electrophoresis on agarose gel containing 0.5% SDS.
3. Results
In untreated samples 3-6, protein DNA migrated faster and was located in the front of the comparison; in the treated samples 3-6, protein DNA and protein are covalently crosslinked and combined to form a complex with larger molecular weight, and the complex is positioned at a sample injection hole and cannot enter gel.
TABLE 2 results of protein-DNA crosslinking test by plasma crosslinking apparatus
EXAMPLE 5 crosslinking apparatus of plasma crosslinking test of proteins and DNA
1. Materials: histone (company, worthington, lot 34E 7307N) and pUC18 plasmid DNA (company Thermo fisher scentific, lot 00134262).
2. The method comprises the following steps: after histone (0.1 mg/ml) and pUC18 plasmid DNA (0.1 mg/ml) are mixed, polytetrafluoroethylene is selected as an insulating medium material, and a mixed gas of helium and air is adopted as working gas to discharge to generate low-temperature plasma:
sample 7: treating the mixture sample for 1min respectively to obtain;
sample 8: treating the mixture sample for 2min respectively to obtain the mixture;
sample 9: treating the mixture sample for 3min respectively to obtain;
sample 10: treating the mixture sample for 4min respectively to obtain;
and adding the sample into a 96-well plate, placing the 96-well plate on a sample placing area to be treated in the cavity, and applying voltage to the power supply to excite the electrode to discharge so as to generate plasma for treatment.
The treated samples were mixed with loading buffer and subjected to electrophoretic analysis in agarose gel (HyAgarose, lot 14150906029) containing 0.5% SDS (Bio-rad, lot L1610302).
3. Results:
in the untreated sample, pUC18 plasmid DNA migrated faster and was located in the front of the comparison; when the treatment is carried out for 1min, the proteins are covalently crosslinked and combined with the DNA, the mobility of pUC18 plasmid DNA is slowed down, and the mobility is different due to the different amounts of the covalently-combined proteins on the DNA, so that a diffuse strip is formed. With the increase of the treatment time, more and more pUC18 plasmid DNA-bound proteins are treated for 2min, the formed complex is bigger and bigger, the mobility is slower, and the complex which cannot enter into gel is started to be formed. At 3 or 4 minutes of treatment, pUC18 plasmid DNA was bound to the protein in its entirety to form a large complex, which could not be electrophoresed into an agarose gel. As can be seen from fig. 5, the present application has an obvious treatment effect and a high treatment efficiency, and the effect is positively correlated with the treatment time.
EXAMPLE 6 crosslinking apparatus of plasma crosslinking test of DNA
1. Materials: pUC18 plasmid DNA (company Thermo fisher scentific, lot number Lot 00134262).
2. The method comprises the following steps: pUC18 plasmid DNA (0.5 mg/ml), polytetrafluoroethylene is selected as an insulating medium material, and mixed gas of helium and air is adopted as working gas to discharge to generate low-temperature plasma:
sample 11: treating the mixture sample for 1min respectively to obtain;
sample 12: treating the mixture sample for 2min respectively to obtain the mixture;
sample 13: treating the mixture sample for 4min respectively to obtain;
the treated samples were mixed with loading buffer and subjected to electrophoretic analysis in agarose gel (HyAgarose, lot 14150906029) containing 0.5% SDS (Bio-rad, lot L1610302).
3. Results:
referring to Table 2, in untreated samples, DNA migrates faster, being located in a position before comparison; in the treated samples 11 to 13, the DNA was covalently cross-linked to the DNA to form a complex.
TABLE 3 crosslinking test results of plasma crosslinking apparatus on DNA
Example 7 crosslinking apparatus of plasma treatment solution for sample crosslinking
1. And (3) material treatment:
sample 14: the method comprises the steps of collecting microorganism or suspension cell culture, suspending in physiological saline, and centrifuging again;
sample 15: the adherent cell culture is treated by pouring out the culture solution in the adherent cell culture and washing with physiological saline for 1-2 times;
sample 16: the animal and plant tissue is cut into thin layers with the thickness of 1-2mm, and the thin layers are placed in a plate to be treated;
sample 17: an extracellular sample, the treating step comprising mixing extracellular proteins, nucleic acids, or both.
2. The method comprises the following steps: physiological saline is selected as a treatment solution, polytetrafluoroethylene is selected as an insulating medium material, and mixed gas of helium and oxygen is adopted as working gas to discharge to generate low-temperature plasma:
and placing physiological saline solution on a sample placing area to be treated in the cavity, applying voltage to the power supply to excite the electrode to discharge so as to generate plasma for treatment, and adding the treated physiological saline solution into the sample.
The genomic DNA of samples 14-16 was extracted using phenol-chloroform extraction. The treated culture or tissue was collected by centrifugation, suspended in physiological saline, added with an equal volume of phenol-chloroform, thoroughly mixed, centrifuged, and the supernatant was aspirated, and the supernatant was mixed with a loading buffer and subjected to electrophoresis on agarose gel containing 0.5% SDS for electrophoresis analysis. Sample 17 was directly mixed with loading buffer and subjected to electrophoresis on agarose gel containing 0.5% SDS.
4. Results
Referring to Table 3, in untreated samples 14-17, protein DNA migrated faster and was located in the front of the comparison; in the treated samples 14-17, protein DNA and protein are covalently crosslinked and combined to form a complex with larger molecular weight, and the complex is positioned at a sample injection hole and cannot enter gel.
Table 4 crosslinking apparatus of plasma treatment solution for sample crosslinking experiment results
Example 8 comparison of UV Cross-Linked Effect
1. Materials: histone (company, worthington, lot 34E 7307N) and pUC18 plasmid DNA (company Thermo fisher scentific, lot 00134262).
2. The method comprises the following steps:
sample 18: pUC18 plasmid DNA (0.1 mg/ml), polytetrafluoroethylene is selected as an insulating medium material, mixed gas of helium and air is adopted as working gas to discharge and generate low-temperature plasma, and the mixture sample is processed for 2min to obtain the high-temperature plasma;
sample 19: mixing histone (0.1 mg/ml) with pUC18 plasmid DNA (0.1 mg/ml), selecting polytetrafluoroethylene as an insulating medium material, adopting a mixed gas of helium and air as working gas to discharge and generate low-temperature plasma, and processing a mixture sample for 2min to obtain the plasma;
control 1: pUC18 plasmid DNA (0.1 mg/ml), 254 nm UV lamp was used, power was about 200 mW/cm 2 Treating the mixture sample for 10 min to obtain the product;
control 2: mixing histone (0.1 mg/ml) with pUC18 plasmid DNA (0.1 mg/ml), and using 254 nm ultraviolet lamp with power of about 200 mW/cm 2 Treating the mixture sample for 10 min to obtain the product;
the treated samples were mixed with loading buffer and subjected to electrophoretic analysis in agarose gel (HyAgarose, lot 14150906029) containing 0.5% SDS (Bio-rad, lot L1610302).
3. Results:
TABLE 5 comparative analysis of plasma crosslinking and UV crosslinking experiment results
The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the application can be made without departing from the spirit of the application, which should also be considered as disclosed herein.
Claims (10)
1. A plasma-based crosslinking apparatus, comprising:
a reaction cavity (1),
a plasma generating device (2) and a sample placing area (3) to be treated are arranged in the reaction cavity (1), and the plasma generating device (2) generates plasma to enable proteins and proteins, nucleic acid and/or proteins and nucleic acid in the sample to be treated to be subjected to covalent crosslinking;
the reaction cavity (1) further comprises a bottom plate, a top cover, a front cover, a rear cover, a left side plate and a right side plate which are perpendicular to the bottom plate, and the bottom plate, the top cover, the front cover, the rear cover, the left side plate and the right side plate enclose a closed cavity; the bottom plate, the top cover, the front cover, the rear cover, the left side plate and/or the right side plate are/is provided with an air inlet (4) and/or an air outlet (5);
the plasma generating device (2) comprises a plate-shaped high-voltage electrode (9), an insulating medium plate (6) and a net-shaped grounding electrode (7), wherein the plate-shaped high-voltage electrode (9) is arranged at the uppermost layer, the insulating medium plate (6) is arranged in the middle, the net-shaped grounding electrode (7) is arranged at the bottom layer and faces the inside of the cavity of the reaction cavity (1), and the plate-shaped high-voltage electrode (9) is connected with a high-voltage line (8);
wherein,,
the plate-shaped high-voltage electrode (9), the insulating dielectric plate (6) and the net-shaped grounding electrode (7) are laminated together in three layers to serve as a top cover of the reaction cavity;
when the device works, mixed gas of air and helium under different proportions is added from an air inlet (4), the introduced gas is ionized at a plasma generating device (2) to generate plasma, active particles in the plasma are diffused into a cavity of a reaction cavity (1), and a biological sample in a sample placing area (3) to be treated is treated;
the sample placing area (3) to be treated comprises a lifting table (10), and the distance between the sample to be treated and the plasma generating device (2) is adjusted by changing the height of the lifting table (10);
the plasma is low-temperature plasma;
changing the height of the lifting table (10) to enable the distance between the sample to be processed and the plasma generating device (2) to be 0.5-2cm;
the crosslinking device further comprises a gas flow controller module (11) connected with the reaction cavity (1), wherein the gas flow controller module (11) provides reaction gas for the plasma generating device (2);
the gas flow controller module (11) comprises a controller (12), a gas inlet (13) and a gas outlet (14), wherein the controller (12) is controlled by a computer to adjust the required working gas types, gas flow and occupied proportion, and the gas outlet (14) is connected with a plasma crosslinking device;
the reaction gas provided by the gas flow controller module (11) is a mixed gas of air and helium.
2. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the plate-shaped high-voltage electrode (9) can adjust discharge frequency and voltage; the voltage range of the plate-shaped high-voltage electrode (9) is between 1kV and 20kV, the frequency range is less than or equal to 100kHz, and the plate-shaped high-voltage electrode is one of pulse, direct current, alternating current and radio frequency power sources.
3. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the nucleic acid is DNA and/or RNA;
the protein is any one or more proteins in the biological sample.
4. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the plate-shaped high-voltage electrode (9) is made of red copper or stainless steel.
5. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the insulating medium plate (6) is made of polytetrafluoroethylene, ceramic, rubber, glass fiber reinforced plastic, crosslinked polystyrene, organic glass, epoxy resin, polyethylene, polycarbonate, quartz glass, mica plate or polyimide.
6. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the net-shaped grounding electrode (7) is made of alloy steel.
7. The plasma-based crosslinking apparatus of claim 1, wherein the plasma-based crosslinking apparatus comprises,
the sample to be treated is a solid biological sample and a liquid biological sample;
the solid biological sample or the liquid biological sample is one or more of microorganism bacterial liquid, cell culture, animal and plant tissues, nucleic acid extract, protein extract, in-vitro protein and nucleic acid mixture.
8. The plasma-based crosslinking apparatus of claim 1, wherein: the device also comprises a timing function module and/or a voltage selection function module, wherein the timing function module can set the processing time, and the voltage selection function module can set different voltages and adjust the discharge power.
9. A method of using a plasma-based crosslinking apparatus according to any one of claims 1 to 8, comprising the steps of:
the first step: processing the material to obtain a sample to be processed;
and a second step of: placing a sample to be treated in a sample placing area (3) to be treated of a reaction cavity (1) of the crosslinking device;
and a third step of: a plasma generating device (2) for generating plasma;
fourth step: covalent cross-linking of proteins with proteins, nucleic acids with nucleic acids, and/or proteins with nucleic acids in the sample to be treated;
wherein, in the first step:
(1) The material is a microbial bacterial liquid or a suspension cell culture, and the processing step comprises the steps of collecting the microbial bacterial liquid or the suspension cell culture, suspending the microbial bacterial liquid or the suspension cell culture in normal saline, pouring the physiological saline into a plate to form a thin layer with the thickness of 1-2mm, and obtaining a sample to be processed; or (b)
(2) The material is an adherent cell culture, and the treatment step comprises pouring out a culture solution in the adherent cell culture, washing with physiological saline for 1-2 times, and then covering with 1-2mm physiological saline to obtain a sample to be treated; or (b)
(3) The material is animal and plant tissue, and the treatment step comprises the steps of cutting the animal and plant tissue into thin layers with the thickness of 1-2mm, and placing the thin layers into a plate to obtain a sample to be treated; or (b)
(4) The material is an extracellular sample, and the processing steps comprise adding extracellular protein, nucleic acid or a sample obtained by mixing the protein and the nucleic acid into a plate to form a thin layer with the thickness of 1-2mm, so as to obtain a sample to be processed;
wherein the time for covalent crosslinking is 1-5min.
10. Use of a plasma-based crosslinking apparatus according to any one of claims 1 to 8 in the field of biology;
wherein the biological domain is a biological domain.
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| CN103611184A (en) * | 2013-11-27 | 2014-03-05 | 长春吉原生物科技有限公司 | Hydrogel of composite semipermeable membrane and preparation method thereof |
| JP2016175895A (en) * | 2015-03-06 | 2016-10-06 | 国立大学法人東北大学 | Plasma-treated protein aqueous solution, protein fixed surface excellent in cell and tissue engraftment properties which is produced using the same, and applications thereof |
| CN105246241A (en) * | 2015-10-30 | 2016-01-13 | 西安交通大学 | Apparatus for generating large-area cold plasma |
| CN105548568A (en) * | 2016-01-27 | 2016-05-04 | 中国人民解放军第三军医大学 | Method for detecting deoxyribonucleic acid (DNA)-protein cross link based on enzyme-linked immuno sorbent assay (ELISA) |
| CN206902147U (en) * | 2017-01-19 | 2018-01-19 | 西安交通大学 | A kind of crosslinking apparatus based on plasma |
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Inventor after: Guo Li Inventor after: Liu Dingxin Inventor after: Zhao Yiming Inventor after: Li Qiaosong Inventor before: Guo Li Inventor before: Liu Dingxin Inventor before: Zhao Yiming Inventor before: Li Qiaosong Inventor before: Kong Gangyu |