Research Project
9048453
? DESCRIPTION (provided by applicant): Analysis of single cells, or cells that are not present in large numbers in the body, is important to understanding basic biology and diseases. A specific example is cancer, in which changes in DNA in a single cell can lead to that cell reproducing rapidly. Similarly, studying the proteins that make up these cells can tell us how the changes in DNA result in changes in cells. The structure can gives us specific targets for therapies, such as chemicals that interfere with the binding of different proteins to one another. To measure the contents of single cells, they must be isolated, broken open (lysed) to release their contents, and measured. When samples are moved from tube to tube, small amounts of liquid are lost, and cells can stick to the surfaces of the vessels. Also, the volumes used are larger than that of a cell, so once the cell is lysed its contents get diluted. Combining isolation, lysis, and detectin, without liquid transfers, results better sensitivity. We would like to create a cell lysis method tat can be used with small samples. Adaptive Focused Acoustics (AFA) focuses ultrasound into a small volume. It works well with for the analysis of proteins and DNA in larger volumes. But closed systems of tubing, flowing the sample from step to step in its analysis, are not usually used. We will use AFA with such a closed system to prevent sample loss and dilution. We will examine use different frequencies of ultrasound, which can focus the sound to a small volume as well as cause more fluctuations in the pressure to break the cells the part of the system put into the AFA will include cell traps made by creating plugs inside tubing. That way the AFA can be applied as the cells sit on the trap. The traps will also be used as microreactors. For understanding proteins, these contain enzymes that break down proteins into components. The mechanism that lyses cells with ultrasound is called cavitation: Little bubbles of form in response to the pressure pulses caused by the ultrasound, they oscillate and collapse, causing turbulent motion. Part of the project will look at ways to make cavitation happen much more frequently. One way is to modify the inside of the tubes (capillaries) that are used, by making them rough or adding particles to them. The presence of these structures gives bubbles a surface to form on. Another method is to make small chambers which have one wall that has structure. To do this we will drill small holes with a laser into materials and seal them into chambers. We will measure whether more cavitation happens with these devices using either high-speed microscopy to see the bubbles or an ultrasound sensor which can hear the sound of bubbles collapsing. Finally, we will measure the efficiency of the devices we make by measuring the proteins and DNA released by different numbers of cells, starting from large numbers of tens of thousands and moving to 1 cell.
