Summary of the invention
The purpose of this invention is to provide a kind of easy and simple to handle, do not cause the sample crossed contamination, can handle the ultrasonic degradation cell of micro-example or shear the macromole device.
Ultrasonic degradation cell provided by the invention or shearing macromole device comprise: form the ultrasonic transducer of focusing acoustic field and the sampling receptacle supporting structure of carry sample container patchhole; Described ultrasonic transducer and described sampling receptacle supporting structure form an enclosed cavity, and ultrasonic transducer constitutes the part of enclosed cavity bottom at least.
Described device also comprises the ultrasonic transducer supporting structure; This supporting structure is positioned at the bottom of described ultrasonic transducer.
Described ultrasonic transducer is spherical concave surface or elliposoidal concave surface ultrasonic transducer with focusing function, have the pipe shape ultrasonic transducer of focusing function or have the circular dull and stereotyped ultrasonic transducer that is made of the Fresnel ring electrode of focusing function.Described ultrasonic transducer supporting structure is the pedestal of tubulose pedestal or other hollow structures.Described ultrasonic transducer should be the part of enclosed cavity bottom at least.In some cases, ultrasonic transducer constitutes the whole bottom of enclosed cavity.
Any suitable sampling receptacle supporting structure all can be used for making up shear of the present invention.The sampling receptacle supporting structure can be a sample hose support or a blocks that is processed with cavity configuration.Preferably a kind of sample hose support of being furnished with a plurality of bases, these bases be detachable and replacement to adapt to different sample volumes.
Any suitable liquid coupling medium all can be used for making up shear of the present invention.Liquid coupling medium can be a kind of high boiling liquid such as water.The liquid level of liquid coupling medium should be as far as possible near the focal plane of focusing acoustic field.The liquid level of preferably liquid couplant is in the focal plane of focusing acoustic field.
Any sampling receptacle all can be used for making up shear of the present invention.Such as, sampling receptacle can be tubular vessel such as test tube or centrifuge tube.Sample can be any suitable volume, such as the sample volume in 1 microlitre to the 10 milliliter scope.
The bottom of sampling receptacle should be as far as possible near the focal plane of focusing acoustic field.The bottom of general sampling receptacle should be positioned on the liquid coupling medium liquid level 0~5 millimeter scope.
Any suitable ultrasonic-frequency power supply all can be used among the present invention ultrasonic in order to produce.Preferably ultrasonic-frequency power supply is adjustable at power with on action time.In addition, preferably the operating frequency of ultrasonic-frequency power supply equals or as far as possible near Frequency for Ultrasonic Energy Transducer.
Shear of the present invention can be worked in any suitable manner, as continous way or pulsed.
Second purpose of the present invention provides a kind of ultrasonic degradation cell or shears macromolecular method.
A kind of ultrasonic degradation cell or shear macromolecular method, this method comprises:
1), and its liquid level is in or the place, focal plane of the focusing acoustic field that forms near ultrasonic transducer at the ultrasonic degradation cell or shear carrying liquid couplant in the enclosed cavity of macromole device;
2) will contain and remain the cracking target cell or wait to shear the macromolecular liquid sample splendid attire of target in sampling receptacle, and the sampling receptacle bottom is positioned at or the place, focal plane of the focusing acoustic field that forms near ultrasonic transducer;
3) ultrasonic transducer is connected on the ultrasonic-frequency power supply to produce focus supersonic, the focusing acoustic field of generation points to contain and remains the cracking target cell or wait to shear the macromolecular liquid sample of target;
In said process, described ultrasonic degradation cell or shearing macromole device comprise: form the ultrasonic transducer of focusing acoustic field and the sampling receptacle supporting structure of carry sample container patchhole; Described ultrasonic transducer and described sampling receptacle supporting structure form an enclosed cavity, and ultrasonic transducer constitutes the part of enclosed cavity bottom at least.
A concrete application of present method is the cracking target cell.Any suitable target cell all can utilize present method to carry out cracking.Typical target cell comprises zooblast, vegetable cell, fungal cell, bacterial cell, artificial recombination cell or culturing cell.Zooblast, vegetable cell, fungal cell, bacterial cell can be taken from any kind or the subspecies of animal kingdom, vegitabilia, mycota or bacterium circle.Take from ciliate, cellular type slime-fungi, flagellate and any kind of microsporozoite or also available present method of cell of subspecies carry out cracking.Take from birds such as chicken, vertebrates class such as fish, all available present method of cell of mammal such as mouse, rat, rabbit, cat, dog, pig, milk cow, bull, sheep, goat, horse, monkey and other inhuman primate and people is carried out cracking.
For zooblast, take from all available present method of cell of a certain particular organization or organ and carry out cracking.As can be used for analyzing the cell of reticular tissue, epithelium, muscle tissue or nervous tissue.Equally, take from all available present method of cell of following organ and carry out cracking, as the eye accessory organ, ring spigot official, ear's organ, the chievitz's organ, the circumventricular organ official, the organ of Corti, vitals, enamel organ, the terminal organ, female genitalia official, the male genitalia official, organ floats, Luo Fenni spray shape organ, reproductive organ, Golgi tendon organ, gustatory organ, the auditory organ, female internal reproductive organ, male internal reproductive organ, (insect) adeagus, Jacobson's organ, the neurohemal organ, the goigi tendon organ official, olfactory organ, the otolithic organ, sagging organ, the organ of Rosenmuller official, sensory organ, the spiral organ, duplicate invoice connects organ, little fornix organ, extra organ, target organ, tactile organ, urinary organ, the organum vasculosum of lamina terminalis official, vestibular organ, vestibulocochlear organs, vestige, the visual organ, vomero nasal organ, the migration organ, Webers' organ official and glomus aorticum organ etc.Treat that preferably the cracked cell is to take from animal body internal such as brain, lung, liver spleen, marrow, thymus gland, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall-bladder, stomach, intestines, testis, ovary, uterus, rectum, neural system, body of gland, internal blood vessel etc.In addition, take from the equal cleavable of cell of any plant materials, fungi such as yeast, bacterium such as eubacterium or archeobacteria.The reconstitution cell of taking from any eucaryon or prokaryotic organism body (as animal, plant, fungi or bacterium) is cleavable also.Be present in the also cleavable of cell in body fluid (as blood, urine, saliva, marrow, seminal fluid or other ascites class fluid) and some separated component (as serum or blood plasma) thereof.
Present method is particularly suited for the cell that cracking has cell walls, as vegetable cell, fungal cell and bacterial cell.
The target organism macromole is promptly sheared in the concrete application of another of present method.Any suitable biomacromolecule all can utilize present method to shear.Can shear DNA or RNA as present method.Any nucleic acid comprises all available present method shearing of strand, two strands and three chain nucleic acid.Above-mentioned nucleic acid comprises DNA (as A-, B-or Z-shape DNA) and RNA (as mRNA, tRNA and rRNA).
The focusing acoustic field that ultrasonic degradation cell of the present invention or shearing macromole device and system thereof produce also can produce at regional area (burnt territory) under the lower-wattage condition be enough to realize brokenly born of the same parents' strong ultrasonic cavitation effect, can carry out fragmentation and shearing treatment to small amounts of cells and biomacromolecule (such as nucleic acid) sample.Because this device power demand is less relatively, and need not to change parts in the operation, is fixed for the ultrasonic-frequency power supply acoustic load, therefore lower to the ultrasonic-frequency power supply requirement, make simple relatively.And this device is not subjected to sample hose shape and volume restrictions, can be directly used in the processing of micro-cell and nucleic acid samples.
Ultrasonic degradation cell of the present invention or shear the macromole device and system replaces the broken born of the same parents' device of traditional probe type ultrasonic with the specified shape ultrasonic transducer that produces focusing acoustic field has crossed contamination simple in structure, no, easy and simple to handle, with low cost, volume I is integrated into advantages such as microsystem.
Embodiment
Embodiment 1, ultrasonic degradation cell or shearing macromole device
As shown in Figure 1, ultrasonic degradation cell of the present invention or shearing macromole device comprise 3 and adjustable ultrasonic-frequency power supplies of sampling receptacle supporting structure (sample hose support) of a spherical concave surface ultrasonic transducer 1, ultrasonic transducer supporting structure (tubular base) 2, carry sample container patchhole.Spherical concave surface ultrasonic transducer 1 supports by a tubular base 2, concave surface is placed up, tubular sample hose support 3 in order to the placement sample hose is fixed on the base 2 by the viscosity waterproof glue, the concave surface of sample hose support 3 and spherical concave surface ultrasonic transducer 1 constitutes the enclosed cavity (container) of carrying liquid couplant 4, and wherein be advisable in the focal plane of the liquid level subglobular concave surface ultrasonic transducer 1 of liquid coupling medium 4; In order to be applicable to the different volumes sample, a disconnectable base 5 is combined into the support that can place the little centrifuge tube 6 of different model with sample hose support 3, the bottom of its medium and small centrifuge tube 6 should place liquid coupling medium 4 liquid levels up and down in 5 millimeters scopes, to reach preferable broken born of the same parents' effect.Treat broken cell suspension 7 splendid attires in little centrifuge tube 6, an adjustable ultrasonic-frequency power supply 8 is connected with spherical concave surface ultrasonic transducer 1 by lead 9, and it is ultrasonic to drive its emission.
Embodiment 2, cracking intestinal bacteria
(1), the intestinal bacteria equal-volume branch of 400 μ L incubated overnight installs in two miniature centrifuge tubes of 0.5mL, centrifugal 60 seconds, abandons supernatant.Every then pipe adds the resuspended thalline of 50 μ l deionized waters respectively, the vibration mixing.
(2), an above-mentioned wherein pipe placed on the sample hose support of the ultrasonic broken born of the same parents' device of embodiment 1 spherical concave surface focus type (as shown in Figure 1), regulating the ultrasonic-frequency power supply output voltage is 35Vpp, opens the ultra-sonic generator power supply, carries out 30 seconds ultrasonic broken born of the same parents and handles;
(3), all centrifugal 2 minutes of above-mentioned each the pipe bacterium liquid that will handle and handle without ultrasonic broken born of the same parents through ultrasonic broken born of the same parents (13,400rpm), shift supernatant then respectively to new centrifuge tube, and add the abundant mixing of isopyknic phenol/chloroform;
(4), with above-mentioned two centrifugal 6 minutes of mixed solutions of pipe (13,400rpm), and shift the upper strata water respectively to new centrifuge tube;
(5), from above-mentioned two centrifuge tubes, get 8 μ l sample liquid respectively and carry out the agarose gel electrophoresis detection.
Electrophoresis result is as shown in Figure 2: swimming lane A-DNA marker (λ DNA/Hinder III); The sample that swimming lane B-handles without ultrasonic broken born of the same parents; The sample that swimming lane C-handles through the ultrasonic broken born of the same parents' device of spherical concave surface focus type.Electrophoresis result shows that intestinal bacteria after the ultrasonic broken born of the same parents' device of spherical concave surface focus type is handled, can be detected the nucleic acid releaser.Prove the ultrasonic broken born of the same parents' device cleavable intestinal bacteria of spherical concave surface focus type.
Embodiment 3, cracking yeast
(1), get incubated overnight centrifugal 1 minute of yeast bacterium liquid two pipe (200 μ l/ pipe) (10,000rpm), abandon supernatant;
(2), above-mentioned two pipes are respectively with the broken resuspended thalline of bacterium liquid of 50 μ l, vibration mixing;
(3), an above-mentioned wherein pipe placed on the sample hose support of the ultrasonic broken born of the same parents' device of embodiment 1 spherical concave surface focus type (as shown in Figure 1), regulating the ultrasonic-frequency power supply output voltage is 35Vpp, opens the ultra-sonic generator power supply, carries out 30 seconds ultrasonic broken born of the same parents and handles;
(4), all centrifugal 2 minutes of the two pipe bacterium liquid that will handle and handle without ultrasonic broken born of the same parents through ultrasonic broken born of the same parents (13,400rpm), shift supernatant then respectively to new centrifuge tube, and add the abundant mixing of isopyknic phenol/chloroform;
(5), with above-mentioned two centrifugal 6 minutes of mixed solutions of pipe (13,400rpm), and shift the upper strata water respectively to new centrifuge tube;
(6), from above-mentioned two centrifuge tubes, get 8 μ l sample liquid respectively and carry out the agarose gel electrophoresis detection.
Electrophoresis result is as shown in Figure 3: swimming lane a-DNA marker (λ DNA/Hinder III); The yeast sample that swimming lane b-handles without ultrasonic broken born of the same parents; The yeast sample that swimming lane c-handles through the ultrasonic broken born of the same parents' device of spherical concave surface focus type.Electrophoresis result shows that yeast after the ultrasonic broken born of the same parents' device of spherical concave surface focus type is handled, can be detected the nucleic acid releaser.Prove the ultrasonic broken born of the same parents' device of spherical concave surface focus type in conjunction with broken bacterium liquid rapidly cracking have the yeast of tough and tensile cell walls.
Reagent: broken bacterium liquid (1%SDS, 50mM EDTA, 0.1M Tris; PH8.0)
Embodiment 4, shearing λ DNA
(1), gets the λ DNA sample liquid that 10 μ l concentration are 0.12 μ g/ μ l with the little centrifuge tube of 0.8ml, and little centrifuge tube is placed on the support of the ultrasonic broken born of the same parents' device of embodiment 1 spherical concave surface focus type (as shown in Figure 1), regulating the ultrasonic-frequency power supply output voltage is 35Vpp, open ultrasonic-frequency power supply, carry out 5 minutes ultrasonic shearing treatment;
(2), from above-mentioned centrifuge tube, get 8 μ l sample liquid and the undressed λ DNA of equivalent sample liquid and carry out the agarose gel electrophoresis detection simultaneously.
Electrophoresis result is as shown in Figure 4: swimming lane A '-DNA marker (λ DNA/Hinder III); The λ DNA of swimming lane B '-handle without ultrasonic broken born of the same parents; The λ DNA that the ultrasonic broken born of the same parents' device of the spherical concave surface focus type of swimming lane C '-process is handled; Swimming lane D '-DNA marker (DL2000, Takara).Electrophoresis result shows that λ DNA after the ultrasonic broken born of the same parents' device of spherical concave surface focus type is handled 5 minutes, almost completely is degraded, and proves that the ultrasonic broken born of the same parents' device of spherical concave surface focus type can shear long-chain DNA quickly and effectively.