Centrifugal bursting

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Osmotic lysis consists of suspending cells in a solution of relatively high ionic strength. This causes water inside the cell to diffuse out through the membrane. The cells are then isolated by centrifugation and transferred to pure water. Water rapidly diffuses into the cell, bursting the membrane. 

Lyse the cells using a sonicator at 80% power using 15 s bursts with 45 s intervals between bursts. Repeat this five times, keeping the cells on ice. Transfer the lysate to centrifuge tubes and pellet the cell debris by centrifugation in a JA-20 rotor (Beckman) for 30 min at 35,000Xy. Immediately transfer the supernatant to a fresh beaker or tube. 

FIGURE 3.29 A schematic view from above the disk of a passive capillary burst valve. A liquid flows in a channel or capillary and is pinned at the discontinuity where the channel meets a chamber or a wider channel. Sufficient fluidic pressure must be exerted by the centrifugal pump to overcome the pressure of curved liquid surfaces and to wet the walls of the chamber with liquid. This pressure is achieved at a characteristic rate of rotation or burst frequency, C0c, above which the liquid exits the channel and enters the chamber. CO, depends on the hydraulic diameter (dH) of the capillary and the amount of liquid in the channel and therefore provides a means of gating the flow of liquid [1042]. Reprinted with permission from the American Chemical Society. 

Passive capillary burst valves were fabricated on a plastic disk in which centrifugal liquid pumping was used to transport fluids, as shown in Figure 3.29. When the angular velocity (co) is less than the critical value (coc), the liquid cannot enter the big chamber. When co > coc, the liquid then bursts into the chamber. A wider channel would require less force to burst through and hence a lower rotation rate or a lower centrifugal force was used for the valve to burst open [226,453,454,1042],... 

This did away with centrifuging. However, possible effects could be that the volume of serum (or plasma) produced from the whole blood sample may be too low, or the erythrocytes may burst or may rub on and haemolyse the sample. Comparison of the methods of the Reflotron system vs. conventional analysis showed that this kind of serum (plasma) separation does not influence the routine methods in clinical chemistry. 

Madou et al. from the University of California, Irvine showed a series of capillary valves to perform enzyme-linked immunosorbent assays (ELISAs) on the centrifugal platform [222], The different assay liquids are held back in reservoirs coimected to the reaction chamber via valves of different burst frequency. The capillary valves are opened subsequently by increasing the frequency of rotation. It was shown that in terms of detection range the centrifugally conducted assay has the same performance as the conventional method on a 96-well plate, but with less reagent consumption and shorter assay time. 

The anacardic acids in the natural CNSL of the raw nut shell are decarboxylated and the resultant cardanol liberated supplements the hot technical CNSL in the bath through the bursting of the outer shell and, after an ideally short period, flows continuously out of the system. The spent processed nuts with the inner shell still intact also pass out of this system and after removal of the superficial CNSL by centrifugation or adsorption are shelled by an automatic procedure in Brazilian and East African technology or manually in Indian practice. 

Enzymatic Assay Cells were harvested by centrifugation at 10,000xg for 10 min. The pellet was washed and suspended in buffer (100 mM phosphate buffer, 1 mM EDTA, 5 mM 3-mercaptoethanol [pH 7.0]). The suspended cells were mixed with glass beads (Sigma), vortexed at maximum rate in bursts of 30 to 120 s, and then cooled on ice. The cell debris... 

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