Cell disruption refers to the technique of destroying the cell membrane and cell wall by external force, and releasing the cell contents including the target product component, and is the basis for separating and purifying the non-secretory biochemical substance (product) synthesized in the cell. Combined with significant advances in recombinant DNA technology and tissue culture technology, proteins previously thought to be difficult to obtain are now available for mass production. However, due to the different composition of bacteria, yeast, fungi, and plant cell walls, and the network structure of the same type of cells is different, the cell wall is different in firmness. Although animal cells do not have a cell wall, they have a cell membrane, and a certain cell disruption method is required to break the membrane to achieve the purpose of extracting the product. First, the cell disruption method Mechanical method 1.1 Organizational mash The material is made into a thin paste liquid, placed in the cylinder about 1/3 volume, the cover is tightly closed, the governor is first dialed to the slowest position, and after the switch is turned on, the speed is gradually accelerated to the required speed. Generally used in animal tissues, plant fleshy seeds, soft leaf buds, etc., the rotational speed can be as high as 10,000 rpm / M or more. Due to the large mechanical shear of the rotating blade, the preparation of some larger molecules such as nucleic acids is rarely used. 1.2 homogenizer The shredded tissue is placed in a tube, then placed in a mortar and slid back and forth, and moved up and down to crush the cells. The gap between the grinding wheel of the homogenizer and the inner wall of the glass tube is maintained at a distance of a few tenths of a millimeter. The material of the homogenizer can be made of hard plastic, stainless steel, artificial fluorescent resin or the like in addition to glass. This method of cell breakage is higher than the high-speed tissue masher, suitable for small amounts and animal organ tissue. Problems: lumps or filamentous fungi that are more likely to cause clogging, smaller Gram-positive heads and some sub-organelles, which are hard and easy to damage the homogenate valve, and are not suitable for treatment by this method. 1.3 Research and Development It is mostly used for bacteria or other hard plant materials. It is often added with a small amount of quartz sand, glass powder or other abrasive to improve the grinding effect. 1.4 bacterial mill It is an improved grinder with a larger grinding area than the mortar and an outlet at the lower part. In the operation, first adjust the bacteria and grinding powder into a paste, add a teaspoon each time, and grind the bacterial cells completely after grinding for 20-30 seconds. 2. Chemical and Biochemical Law 2.1 autolysis method A method of disrupting cells by using their own enzyme system in a tissue cell at a certain pH and an appropriate temperature. This process takes a long time, and a small amount of preservatives such as toluene, chloroform, etc. are often used to prevent cell contamination. 2.2 Enzymatic dissolution method The cell wall is decomposed by various hydrolases such as lysozyme, cellulase, snail enzyme, hemicellulase, lipase, etc., and the cell contents are released. Some bacteria are not sensitive to lysozyme, and after adding a small amount of sulfhydryl reagent or 8 moles of urea, they are converted to be sensitive to lysozyme and dissolved. 2.3 Chemical infiltration method Certain organic solvents (such as benzene, toluene), antibiotics, surfactants, metal chelators, denaturants and other chemicals can change the permeability of the cell wall or membrane to allow the permeate to selectively permeate. Its mechanism of action; chemical penetration depends on the type of chemical reagent and the structure and composition of the cell wall and membrane. 3. Physical law 3.1 repeated freeze-dissolved method Principle: The cells are destroyed by sudden freezing, the formation of intracellular ice crystals and sudden changes in the concentration of intracellular and extracellular solvents. Method: The cells to be broken are frozen under -20 degrees, melted at room temperature, and repeated several times. The cell structure is broken due to the formation of ice particles in the cells and the increase of the salt concentration of the remaining cell liquid. 3.3 Rapid thermal quenching method The material is placed in boiling water for 85-90 minutes and rapidly cooled in a water bath. This method can be used for bacterial and viral materials. Second, the ultrasonic fracture mechanism Ultrasonic waves are an elastic mechanical wave in a matter medium, which is a form of wave, so it can be used to detect physiological and pathological information of the human body, and to diagnose ultrasound. At the same time, it is a form of energy. When a certain dose of ultrasound is transmitted through the body, the interaction between them can cause changes in the function and structure of the organism, that is, the ultrasonic biological effect. Zhuhai Mingke Electronics Technology Co., Ltd , https://www.zhmkdz-electronics.com
The effects of ultrasound on cells are mainly thermal effects, cavitation effects and mechanical effects.
The thermal effect is that when the ultrasound propagates through the medium, the friction hinders the molecular vibration caused by the ultrasound, converting part of the energy into local high heat (42-43 ° C), because the critical lethal temperature of normal tissue is 45.7 ° C, and the tumor tissue ratio Normal tissue sensitivity is high, so at this temperature, the metabolism of tumor cells is impaired, DNA, RNA, and protein synthesis are affected, thereby killing cancer cells and normal tissues are not affected.
The cavitation effect is that under the irradiation of ultrasound, vacuoles are formed in the living body, and mechanical shear pressure and turbulence are generated along with the vibration of the vacuole and its violent explosion, which causes the tumor to hemorrhage and the tissue to collapse and cause necrosis. In addition, when the cavitation bubble bursts, it generates instantaneous high temperature (about 5000 ° C) and high pressure (up to 500 × 104 Pa), which can thermally dissociate the water vapor to produce .OH radicals and .H atoms, which are composed of .OH radicals and .H. Atomic induced redox reactions can lead to polymer degradation, enzyme inactivation, lipid peroxidation, and cell killing.
The mechanical effect is the primary effect of ultrasound. During the propagation process, the medium particles alternately compress and stretch to form a pressure change, causing damage to the cell structure. The strength of the killing effect is closely related to the frequency and intensity of the ultrasound.
Third, ultrasonic application
1. Ultrasonic extraction of biological nanometer (ultrasonic chemical synthesis method)
In the ultrasonic chemical reaction, the key effect is the cavitation effect of the acoustic wave. During the irradiation of the ultrasonic wave, cavitation bubbles will form, grow and collapse in the liquid, and a cover will be formed when the cavitation bubble collapses. The strong pressure pulse produces many unique properties, such as producing high temperatures up to 5000K and pressures greater than 200Mpa, which is the source of energy for ultrasonic chemical synthesis, which can be used to synthesize nanoparticles on specific powder surfaces.
2, ultrasonic pharmaceutical
2.1 Dispersion of pharmaceutical substances for injection
The phospholipids are mixed with cholesterol in an appropriate manner and mixed with the drug in an aqueous solution, and ultrasonically dispersed to obtain smaller particles for intravenous injection.
2.2 herbal extract
Ultrasonic dispersion is used to destroy plant tissue, accelerate the penetration of solvent into tissue, and improve the extraction rate of active ingredients in Chinese herbal medicine. For example, all alkaloids in the bark of the cinchona can be invaded by the general method for more than 5 hours, and the ultrasonic dispersion can be completed in half an hour.
2.3 Preparation of suspension
Under ultrasonic cavitation and vigorous agitation, a solid drug is dispersed in an aqueous solution containing a surfactant to form an oral or intravenous suspension of about 1 um. Examples are "intravenous camptothecin suspension", "liver contrast agent", "barium sulfate suspension".
2.4 Preparation of vaccine
After the cells or germs are killed by ultrasonic dispersion, the vaccine is prepared by an appropriate method.
3. Ultrasonic dispersion of cosmetics
In order to further extract the essence of the drug and the micronization of the particles, and to save production costs, to achieve the dispersion and emulsification effect, the cosmetics penetrate deep into the inner layer of the skin, let the skin absorb well, exert the efficacy and effect of the drug, and adopt ultrasonic emulsification. Achieve very good results. By ultrasonic dispersion, fine particles of oil such as wax and paraffin emulsified or lotion can be dispersed without using an emulsifier. Paraffin particles dispersed in water can be up to 1um in diameter.
4, ultrasonic alcoholization of alcohol - aging technology
A bottle of fine wine is mellow, soft and soft, and rich in fragrance. People often use old wine to describe the preciousness of wine. A bottle of wine from the last century is priced at tens of thousands of yuan. The price means the storage of time. . The main controlling factor of wine is the chemical change, ie the formation of acid, and further esterification, the ester is involved in the association of ethanol and water. The wine that has just been shipped contains sterols, which have a spicy taste. It takes a long time to resolve. This slow change is called alcoholization. Ultrasonic treatment with a power of 1.6KW and a frequency of 17.5-22KHZ for 5-10 minutes can shorten the ripening time of wine by 1/3 to 1/2.
Fourth, the advantages and disadvantages of ultrasonic fracture technology
At present, ultrasonic instruments mainly include ultrasonic oscillators and ultrasonic pulverizers.
The advantage is that it maintains cell activity, saves time, has less sample loss, and can handle large numbers of samples. Therefore, the continuous development and improvement of ultrasonic cell pulverizers and the contribution to large-scale production are considerable. Ultrasonic pulverizer for ultrasonic pulverization is currently the most commonly used method of crushing in biological production. Fermentation broth for different strains. The effect of ultrasonication is quite different. In general, bacilli are more easily broken than cocci, and Gram-negative bacterial cells are more easily broken than Gram-positive bacterial cells, and have a poor effect on yeast.
Advantages: easy to operate, low fluid loss, suitable for laboratory scale. Disadvantages: high cost; easy to cause a sharp rise in temperature; in large-scale operation, the transfer of sound energy and heat is difficult, the resulting chemical radicals are easy to inactivate the product, so it affects its application in large-scale industries. .
V. Summary
Ultrasonic cell pulverizers can and have been widely used in the fields of biochemistry, microbiology, physics, physics, zoology, agronomy, medicine, pharmaceuticals, etc.
As a method of cell disruption, ultrasonic disruption is more common in laboratory scale. When handling a small amount of sample, the operation is simple and the liquid loss is small, but the chemical radicals generated by ultrasonic waves can deactivate certain sensitive active substances. Moreover, the sound energy transmission of the large-capacity device is difficult, and the corresponding cooling measures should be taken. It can be seen from the front that the ultrasonic disruption cell technology is far from perfect and needs further development. 