CN101565691A - Method of separating and purifying single virus - Google Patents
Method of separating and purifying single virus Download PDFInfo
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- CN101565691A CN101565691A CNA2008100364286A CN200810036428A CN101565691A CN 101565691 A CN101565691 A CN 101565691A CN A2008100364286 A CNA2008100364286 A CN A2008100364286A CN 200810036428 A CN200810036428 A CN 200810036428A CN 101565691 A CN101565691 A CN 101565691A
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Abstract
The invention provides a method of separating and purifying a single virus, comprising the following steps: (1) placing a virus sample on an even substrate surface; (2) imaging the virus by utilizing an atomic force microscope and obtaining the physical property of the virus; (3) selecting a single virus in an appropriate area according to the physical property; (4) scanning in the area containing the single virus with the needle tip of the atomic force microscope, determining the reduction height of the needle tip to enable the needle tip to contact with the virus according to the physical property when the needle tip scans close to the virus, carrying out the push nanometer operation to enable the virus to be adsorbed on the needle tip through regulating the acting force between the needle tip and the virus by changing the reduction height of the needle tip of the probe, and finally achieving the separation of single virus. The invention can separate a single virus, and can separate by positioning, i.e. the invention can separate specific target virus particles according to different physical properties of a virus such as the shape, the size, the elasticity, and the like. In addition, the invention can carry out the subsequent biochemical analysis of RNA or DNA, and has biological significance and application value.
Description
Technical field
The present invention relates to a kind of isolation and purification method of virus, particularly a kind of method of separating single virus based on the atomic force microscope nanometer manipulation technology.
Background technology
Compartment analysis to biomolecules is the gordian technique and the important content of biological study always.The fast development of life science and biotechnology press on the yardstick of microcosmic more (unicellular or single molecules level) it is separated so that obtain the semiochemical means of associated biomolecule and instrument (university chemistry, 2004,19:10-15).Over past ten years, successively emerged the technology (assay office that can separate trace even single specific organism of all kinds, 2002,21:97-104), mainly comprise flow cytometry (Cytometry, 2000,42:284-289), the micro-separation of laser capture (Science, 1996,274:998-1001), capillary electrophoresis (Journal of Chromatography B, 1999,722:141-151), two-dimensional electrophoresis (Trends Food.Sci.Technol., 1995,6:51-58), light tweezer (Trends Food.Sci.Technol, 1995,6:51-58), the magnetic tweezer (Nature ReviewsMolecular Cell Biology, 2000,1:130-136), micro-fluidic chip (Applied PhysicsLetters, 2004,84:1786-1788).Because technology limitation, these means also are in isolated mononuclear cell or trace molecule mostly at present, its resolving power also has only micro-meter scale, but to remaining the comparison difficulty separating of single virus and single virus, and can not locate separation.
Atomic force microscope (atomic force microscopy AFM) is not only a kind of imaging means with nanometer resolution, and be a kind of can carry out the instrument that nanometer positioning handles (Micron, 2002,33:385-397).People have successively attempted with AFM probe separates trace even single biomolecules.Took the lead in having realized clean cut separation (J Struct.Biol. in 1997 as professor Heckl leader's research group to chromosomal specific region, 1997,119:232-237), but the material that obtains is the mixture that comprises protein and nucleic acid etc., can't all obtain on all four sample at every turn; Afterwards, Oesterhalt etc. with afm tip from the halophilic bacterium purple membrane isolated single protein molecule (Science, 2000,286:143-146), but problem is to want in advance specific antibody that needle point is done modification, its repeatability also has problems; Osada in 2003 etc. are inserted into cell interior with afm tip and separate mRNA, thereby studied gene at intracellular expression, but this is merely able to realize by adherent mode at random, can't accurately locate, more can not guarantee each isolating quantity (Journal of Nanobiotechnology, 2003,1:2).Lu etc. have successfully realized the cutting of the location of single DNA molecules with afm tip and have separated (J.Am.Chem.Soc., 2004,126:11136-11137), but because virus mostly is the particle of protein enclosure bag quilt greatly, diameter arrives hundreds of nanometer size in tens nanometers, more a lot of greatly than protein and DNA, and very frangible, but AFM also of no use at present separates the report of single virus.
Summary of the invention
The technical problem to be solved in the present invention is exactly the limitation that overcomes above-mentioned prior art, provide a kind of can be to the method for single virus isolation and purification.
It is exactly to adopt the method for nano-manipulation technical point from single virus that the separation method of a kind of single virus provided by the present invention is put it briefly.Specifically, this method comprises the following steps:
(1) viral sample is positioned over smooth substrate surface;
(2) utilize imageable tripping device that viral sample is carried out imaging, and obtain its physical properties;
(3) according to the different selected single virus of physical properties;
(4) containing the regional interscan of single virus with the tripping device probe tip, when needle point scanning is neighbouring to virus, physical properties according to the isolating virus of want, reduce needle point to certain altitude, make needle point contact and virus is attached on the needle point, thereby realize the separation of single virus with virus.
Said substrate, the preferably substrate of atomically flating can be a mica, also can be silicon, also can be glass, also can be graphite, can also be the substrate after the chemically modified.
Said viral sample can be a viral solution purified or not purifying, also can be tissue or the cell section that contains virus.
Said single virus can be a virion, also can be naked virus.
Said according to the different selected single virus of physical properties, be that pattern, size, the elasticity according to virus is selected.
Described needle point contact with virus and virus is attached on step on the needle point be nano-manipulation by pushing away, and the height of the reduction of change probe tip is regulated the reactive force between needle point and the virus and realized.
Said separation single virus can be once only to separate a virus, also can be to separate single virus continuously.
Described tripping device is an atomic force microscope or based on the device of atomic force microscope principle.
The probe of said tripping device can be through modification or not modified.
The needle point of the probe of said tripping device is single, also can be probe array, thereby can walk abreast separation.
The probe of said tripping device can separate the sample that is in air, liquid or vacuum environment.
The reduction of said probe tip can also can realize by changing set-point (setpoint) or amplitude size by raising pattern (lift mode).
Said virus can be for different size, different shapeies, have or do not have coating, dna virus or RNA viruses, viroid or virus-like particle.
The present invention can separate single virus, and can position separation.Owing to be that separate the location, just can separate specific virion targetedly according to the difference of physical propertiess such as the character of virus, size, elasticity.And because most viruses are by the RNA of protein bag quilt or the particle of DNA, and break easily, so DNA isolation or proteinic technology are to realize isolating to single virus usually.Also be the RNA or the DNA particle of protein bag quilt just because of most of viruses, the present invention makes and can carry out the generate subsequent fractional analysis to RNA or DNA to have important biological significance and using value to the isolating realization of single virus.
Description of drawings
Fig. 1 is the process synoptic diagram that utilizes the atomic force microscope probe isolated viral.Wherein a has shown the state before separating, and the zone of delineation is for wanting isolating virus, b shown that probe sticks and isolated viral after state.
Fig. 2 utilizes atomic force microscope probe to separate adeno-associated virus: wherein a is the atomic force microscope images before separating; B is the atomic force microscope images after separating.Among the figure, scale is 500 nanometers.
Fig. 3 utilizes atomic force microscope probe to separate ancient bacterium virus: wherein a is the atomic force microscope images before separating; B is the atomic force microscope images after separating.Among the figure, scale is 500 nanometers.
Embodiment
Be that example illustrates method of the present invention with adeno-associated virus, ancient bacterium virus, cucumber mosaic virus, ghost adeno-associated virus emphatically below.
Embodiment 1: the method for utilizing the single adeno-associated virus of atomic force microscope nanometer manipulation technology isolation and purification
Concrete steps are as follows:
1, viral sample is positioned over the substrate surface of atomically flating;
In order to make virus moderate adsorptive power be arranged to satisfy AFM imaging and can isolating needs simultaneously, must select suitable substrate or suitable processing is carried out in substrate according to the character of virus itself at substrate surface.For reaching the purpose of separating adeno-associated virus, we have selected for use mica as solid substrate.
2, with atomic force microscope (AFM) adeno-associated virus is carried out imaging, physical propertiess such as the height of acquisition adeno-associated virus and size;
At first carrying out the adeno-associated virus sample prepares, adeno-associated virus is available from Vector Gene Technology Co., Ltd's (catalog number (Cat.No.): A20115a, name of product: rAAV2-Null), virus is diluted to 107v.g/ μ l with ultrapure water, get the above-mentioned viral drips of solution of 4 μ l and be added on clean sheet mica center, leave standstill and dried up with uncontaminated air or liquid nitrogen in 5 minutes, carry out imaging with AFM.During imaging, adopt AFM to rap pattern (tapping mode), under relative humidity is atmospheric condition under 30 ~ 40%, obtain, see Fig. 2 a.
3, the selected single virus particle in suitable zone;
According to the size (about diameter 20nm) and height of virus, the selected isolating target viral of wanting.For more effective sample separation, preferably select those in substrate, to have only a virus, and in certain distance around it, the zone that does not have other samples to exist.Behind the selected sample, be located the central position in as far as possible little zone, carry out sepn process then.
4, containing the regional interscan of single virus with atomic-force microscope needle-tip, when virus is arrived in the needle point short scan, reduce needle point, needle point is contacted with virus, and carry out the nano-manipulation of " pushing away ", by changing the height that the set-point changes the attenuating of probe tip, thereby regulate the reactive force between needle point and the virus, virus is attached on the needle point, thereby realizes the separation of single virus.
Detailed process is: rap line of mode scanning with AFM earlier, obtain the information (power of this line, monitor and adjust (size that changes scanning forces) when the perhaps height that it converted to), then the same line being carried out contact mode (contact) scanning for the second time thus realize manipulation to this line.Whole imaging and manipulation process need not withdraw from the feedback system of AFM and carry out continuously.Such manipulation can obtain very high accuracy, and this mainly is the restriction that is subjected to the probe yardstick.When carrying out the nano-manipulation of " separation ", obtain piece image, as Fig. 3 a, selected interesting areas; Next, driving the AFM probe by increase power contacts with selection area, thereby utilize moving of AFM probe, and the zone of scanning is as far as possible little, carry out the two-dimensional scan process then, in this scanning process,, overcome substrate and sample transfer is adsorbed onto on the probe the absorption of sample by changing the size of probe to the sample molecule institute application of force, thereby finish the separation of virion, see Fig. 2 b.
The needle point of the probe of atomic force microscope is single in the present embodiment.The AFM imaging of above virus is finished in NANOScope IIIa AFM system with separating all, and carries out under air ambient.Scanner head is the E type.The AFM probe is Force modulation probe (Digital Instrument, the U.S.).Atomic force microscope among the above embodiment is existing commercial apparatus.
Embodiment 2: the method for utilizing the single ancient bacterium virus of atomic force microscope nanometer manipulation technology isolation and purification
Concrete steps are as follows:
1, viral sample is positioned over the substrate surface of atomically flating, selects for use silicon chip herein as solid substrate.
2, with atomic force microscope (AFM) ancient bacterium virus is carried out imaging, obtain the physical properties of ancient bacterium virus;
At first carry out ancient bacterium viral sample and prepare, ancient bacterium virus is extracted from ancient bacterium voluntarily for this laboratory, and is not purified.Above-mentioned viral solution places clean silicon chip center, leaves standstill to dry up with uncontaminated air or liquid nitrogen in 5 minutes, carries out imaging with AFM.During imaging, adopt the AFM contact mode, under relative humidity is atmospheric condition under 30 ~ 40%, obtain, see Fig. 3 a.Two kinds of visibly different viruses of size are arranged on image, and a kind of diameter is 40-50nm, and a kind of is about 120nm, and present embodiment separates the small virus of 40nm.
3, selected diameter is a single virus particle about 80nm in suitable zone;
Be chosen in and have only a virus in the substrate, and the zone that in certain distance around it, does not have other samples to exist.Behind the selected sample, be located the central position in as far as possible little zone, carry out sepn process then.
4, containing the regional interscan of single virus with atomic-force microscope needle-tip, when virus is arrived in the needle point short scan, reduce needle point, needle point is contacted with virus, and carry out the nano-manipulation of " pushing away ", and change the altitude mixture control needle point of attenuating of probe tip and the reactive force between the virus by changing the amplitude size, virus is attached on the needle point, thereby realize the separation of single virus, shown in Fig. 3 b; After having separated a virus, use same needle point, carry out said process once more, separate having obtained second virus.
Detailed process is with embodiment 1.
The needle point of the probe of atomic force microscope is single in the present embodiment.
The AFM imaging of above virus is to finish on Bioscopy (Digital Instrument, the U.S.) with separating, and carries out under vacuum environment.Scanner head is the J type.The AFM probe is silicon probe (Silicon-MDT Ltd., a Russia).Atomic force microscope among the above embodiment is the tripping device that utilizes based on the atomic force microscope principle design.
Embodiment 3: the method for utilizing the single cucumber mosaic virus of atomic force microscope nanometer manipulation technology isolation and purification
Concrete steps are as follows:
1, will contain viral tissue slice and be positioned over smooth substrate surface, select for use at the bottom of the glass chip of polylysine modification herein.The method of suffering from the cucumber leaves tissue slice routinely of mosaic disease is prepared viral sample.
2, with atomic force microscope (AFM) cucumber mosaic virus is carried out imaging, in tissue, find the zone of containing virus; During imaging, adopt the AFM contact mode, in the environment of near vacuum.Can see that from image diameter is the particle about 25nm, has tangible difference with surrounding tissue.
3, selected circle and diameter are single virus particle about 25 nanometers in suitable zone;
Be chosen in and have only a virus in the substrate, and the zone that in certain distance around it, does not have other samples to exist.Behind the selected sample, be located the central position in as far as possible little zone, carry out sepn process then.
4, containing the regional interscan of single virus with atomic-force microscope needle-tip, when virus is arrived in the needle point short scan, reduce needle point, needle point is contacted with virus, and carry out the nano-manipulation of " pushing away ", change the altitude mixture control needle point of attenuating of probe tip and the reactive force between the virus by the size that changes the set-point, virus is attached on the needle point, thereby realizes the separation of single virus.
Detailed process is with embodiment 1.
The needle point of the probe of atomic force microscope is a plurality of in the present embodiment, separates a plurality of viruses simultaneously with different needle points.
The AFM imaging of above virus is to finish on Bioscopy (Digital Instrument, the U.S.) with separating, and carries out under vacuum environment.Scanner head is the E type.The AFM probe is silicon nitride probe (Silicon-MDT Ltd., a Russia).Atomic force microscope among the above embodiment is the tripping device that utilizes based on the atomic force microscope principle design.
Embodiment 4: the method for utilizing the single ghost adenovirus of atomic force microscope nanometer manipulation technology isolation and purification
Concrete steps are as follows:
1, viral sample is positioned over the substrate surface of atomically flating;
In order to make virus moderate adsorptive power be arranged to satisfy AFM imaging and can isolating needs simultaneously, must select suitable substrate or suitable processing is carried out in substrate according to the character of virus itself at substrate surface.For reaching the purpose of separating adeno-associated virus, we have selected for use the mica of modifying with polylysine as solid substrate.
2, with atomic force microscope (AFM) adenovirus is carried out imaging, and obtain the physical properties of virus, the elasticity of ghost virus is less than the virus of non-ghost.
At first carry out ghost adenovirus sample and prepare, (catalog number (Cat.No.): Ad0125d, name of product: Ad5F35-Null), virus is diluted to 10 with ultrapure water to the ghost adenovirus available from Vector Gene Technology Co., Ltd
7V.g/ μ l gets the above-mentioned viral drips of solution of 4 μ l and is added on and modifies sheet mica center, back, leaves standstill to dry up with person's liquid nitrogen in 5 minutes, carries out imaging with AFM.During imaging, adopt AFM to rap pattern (tappingmode), under relative humidity is atmospheric condition under 10 ~ 20%, obtain this viral elastic characteristic.
3, the selected single ghost virion in suitable zone;
Judge elastic difference mutually or measure its elasticity by phase, determine which is a ghost virus by the method for (PhysicalReview E, 71:062901,2005) such as Xingfei Zhou.For more effective sample separation, preferably select those in substrate, to have only a virus, and in certain distance around it, the zone that does not have other samples to exist.Behind the selected sample, be located the central position in as far as possible little zone, carry out sepn process then.
4, containing the regional interscan of single virus with atomic-force microscope needle-tip, when the needle point short scan is viral to ghost, to separate ghost virus because be, want score to reduce 1-2nm more, needle point is contacted with virus, and carry out the nano-manipulation of " pushing away " from solid virus, by changing the height that amplitude changes the attenuating of probe tip, thereby regulate the reactive force between needle point and the virus, virus is attached on the needle point, thereby realize the separation of single virus.
The needle point of the probe of atomic force microscope is single in the present embodiment.The AFM imaging of above virus is finished in NANOScope IV AFM system with separating all, and carries out under air ambient.Scanner head is the E type.The AFM probe is Force modulation probe (Digital Instrument, the U.S.).Atomic force microscope among the above embodiment is existing commercial apparatus.
To other different size, different shapeies, have or do not have coating, dna virus or RNA viruses and also can adopt the method for above-mentioned nano-manipulation based on atomic force microscope to be separated, do not repeat them here.
Claims (15)
1, a kind of isolation and purification method of single virus is characterized in that this method comprises the following steps:
(1) viral sample is positioned over smooth substrate surface;
(2) utilize imageable tripping device that viral sample is carried out imaging, and obtain the physical properties of virus;
(3) according to the selected single virus of physical properties;
(4) containing the regional interscan of single virus with the tripping device probe tip, when needle point scanning is neighbouring to virus, physical properties according to the isolating virus of want, reduce needle point to certain altitude, make needle point contact and virus is attached on the needle point, thereby realize the separation of single virus with virus.
2, method according to claim 1 is characterized in that this substrate is the substrate of atomically flating.
3, method according to claim 1 is characterized in that viral sample is viral solution purified or not purifying or tissue or the cell section that contains virus.
4, method according to claim 1 is characterized in that in the step of selected single virus, and the physical properties of described virus is that pattern, size, the elasticity according to virus is selected.
5, method according to claim 1, it is characterized in that described needle point contact with virus and virus is attached on step on the needle point be nano-manipulation by pushing away, the height of the reduction of change probe tip is regulated the reactive force between needle point and the virus and is realized.
6, method according to claim 1, the separation that it is characterized in that described single virus comprises and once only separates a virus or separate single virus continuously.
7, method according to claim 1 is characterized in that described tripping device is an atomic force microscope or based on the device of atomic force microscope principle.
8,, method according to claim 1 and 2, it is characterized in that this substrate is the substrate after mica, silicon chip, glass, graphite or the chemically modified.
9,, it is characterized in that the single virus in the viral sample is tunicary virion or naked virus according to claim 1 or 3 described methods.
10,, it is characterized in that described viral sample is the sample that is in air, liquid or vacuum environment according to claim 1 or 3 described methods.
11, method according to claim 5, the reduction that it is characterized in that described probe tip is by raising sample, changing set-point or the realization of amplitude size.
12, according to claim 1,5 or 11 described methods, the needle point that it is characterized in that described probe is for the process modification or not modified.
13, according to claim 1,5 or 11 described methods, the needle point that it is characterized in that described probe is single or array.
14. method according to claim 9 is characterized in that described viral sample is the sample that is in air, liquid or vacuum environment.
15, method according to claim 12, the needle point that it is characterized in that described probe is single or array.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831409A (en) * | 2010-05-10 | 2010-09-15 | 杨季芳 | Method for separating marine algae viruses by high-flux tangential flow with virus activity maintaining |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831409A (en) * | 2010-05-10 | 2010-09-15 | 杨季芳 | Method for separating marine algae viruses by high-flux tangential flow with virus activity maintaining |
| CN101831409B (en) * | 2010-05-10 | 2012-07-04 | 浙江万里学院 | Method for separating marine algae viruses by high-flux tangential flow with virus activity maintaining |
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