Cell disruption and lysis by ultrasound

Ultrasonic homogenizers are mainly used for sample preparation and production. These areas include, in particular, the homogenization, emulsification and suspension of various substances, as well as the acceleration of chemical reactions, cell disruption and the extraction of cell contents. Using ultrasonic homogenizers certain substances can be selectively destroyed, tedious preparation processes can be shortened and the yield of many reactions increased. The comparison with mechanical processing devices such as planetary ball mills, rotor / stator or gap homogenizers shows that ultrasonic homogenizers work with higher efficiency and, in particular, make reproducible results possible. The trend in analytics is towards smaller and smaller sample volumes and the reduction in the use of chemicals. For example, the use of ultrasonic homogenizers has become of central importance in recent years, where even the smallest sample quantities are to be processed quickly, cost-effectively and reproducibly.

Disruption of cells and microorganisms

In modern laboratories, ultrasonic homogenizers are used to break cell walls to extract the cell contents, e. g. the proteins without damaging them. A part of the energy introduced into the cell suspension is transformed into heat by friction. In order to avoid thermal damage to the cell contents, the sample is either cyclically intermittent sonicated or cooled in a cooling vessel during sonication. A rosett cell enables a uniform sonication of microorganisms as the ultrasonic energy forces the sample to circulate repeatedly under the probe and through-out the side arms. Placed in the ice bath, the content can be cooled effectively due to the enlarged glass surface.

The disruption of cell membranes depends strongly on the elasticity of the cells. Cell components, such as mitochondria or cytoplasm, can be disrupted fractionally by varying the input ultrasonic energy and thus the extraction power. In the case of particularly resistant bacteria (for example streptococci), fungi, spores, yeasts or tissue samples, direct destruction with very high ultrasonic amplitudes via micro tips is possible, since micro tips can achieve a very large energy input into smallest sample quantities.

When working with microliter quantities, foaming and splashing out of the vessel are a bigger problem. A valuable sample material loss is possible. Therefore the power regulation is very important. If cells with labile walls are to be disrupted, only little power or small amplitude is necessary.

In order to dissolve large quantities continuously, special flow vessels made of glass or stainless steel with a sonication chamber are used to treat each particle of a suspension with the same intensity. Thermal damages to cell contents can be excluded if the vessel is additionally equipped with a cooling jacket. In order to avoid contamination by foreign particles - for example erosion particles of the probe - an indirect sonication in a cup booster or cup horn is to be preferred. This method achieves uniform intensity and cooling.

Applications in biochemistry and medicine:

Disruption of tissue cultures

Subcellular components and viruses are disrupted without any destruction.  

Paternity tests

Rapid extraction of stroma-free hemolysate from the EDTA blood of the putative father for the evaluation of paternity (reduction of the preparation time by approx. 30 min).

Urology

Biochemical membrane analysis on components of spermatozoa. 

Genetic research

Extraction of DNA from human material. 

Liposome Preparation

Disintegration of MLV (Multilamellar Liposomes) using ultrasound (20 kHz) is the predominant method for the production of SLV (Unilamellar liposomes). 

Treatment of smallpox vaccines

Preparation of a uniformly distributed infection solution. 

Dispersing

With ultrasound energy, solid particles or even liquids can be dispersed into another carrier. Nanoscale powders such as titanium dioxide or pyrogenic silica are increasingly used in the production of test paints and lacquers or for the polishing of small body surfaces due to their large specific surface area and the increasing reaction potential. Moreover, these substances have the negative tendency to agglomerate, as a result of which flow and wetting properties deteriorate. The agglomerates formed are destroyed by means of an ultrasonic homogenizer and the dispersion is permanently stabilized against a reagglomeration.

In particle size analysis, dispersing is of great importance for the measurement process. The particles are recognizable in the measuring process only as well as they appear as a detectable measuring signal in the measuring zone. Thus, non-dispersed agglomerates lead to considerable mismeasurements. With the help of ultrasound, the particles are finely divided and thus prepared for a subsequent measurement.

When emulsified with ultrasound, two immiscible liquids such as oil and water are processed into a quasi-homogeneous emulsion. Compared to conventional methods using e. g. rotors, it is possible with the help of ultrasound to produce finely dispersed emulsions with very small droplet sizes and very high stability. Neither clump or cluster formation nor sedimentation of droplets take place. With conventional methods such as rotors or stirrers, slow stirring often results in the separation of the liquid. A too rapid stirring leads to undesirable air inclusions. Ultrasonic homogenizers are often used in pharmacies for the high-quality small-scale production of ointments.

Ultrasonic homogenized emulsions are encountered in many different forms in everyday life, for example in cosmetics or lotions.

Homogenizing

The technical application possibilities of ultrasonic homogenizing range from the production of paints and lacquers to the homogenization of waste water and soil samples for analysis purposes up to sample preparation for grain size analysis. Industrial wastewater, in particular, is continuously examined for the presence of heavy metals, fats or oils in environmental laboratories in order to initiate immediate measures in the event of concentrations being exceeded. For representative analytical results, it is necessary to convert the wastewater samples into a homogeneous state. This is achieved by ultrasonic homogenization within a very short time and with high reliability.

For the characterization of waste samples with regard to their possibility for landfill and the assessment of pollutants, such as PAHs (polycyclic aromatic hydrocarbons), heavy metals or MKW (mineral oil hydrocarbons) in soils, extraction with ultrasound is used as a fast homogenization process as an alternative to elution.

In agriculture, ultrasonic homogenizers, are used for sample preparation to determine subsequently the THC content in hemp as well as in the determination of PAH concentrations in vegetable foods, for example strawberries, depending on the soil load.

Ultrasonic homogenizers are used very frequently in quality control of foodstuffs. To comply with the limit values, the nitrate content of cheese must be determined in the laboratory. The previous method using xylenol methanol distillation and subsequent photometry is toxicologically extremely questionable and particularly time-consuming. For the quantitative determination of the nitrate content, the cheese is therefore alternatively mechanically pre-crushed. This is followed by an intensive and fine homogenization with ultrasound in a rosette cell within a very short time. The achievable particle size is less than 1 μm, and the subsequent filtration for the quantitative washing out of ions is considerably facilitated since no conglomerate formation can take place.

Ultrasonic homogenizer HD 4100