Acoustic lysis and extraction system for limited cell number applications

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 andquot cell trapsandquot 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 andquot microreactorsandquot For understanding proteins these contain enzymes that break down proteins into components The mechanism that lyses cells with ultrasound is called andquot cavitationandquot 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 andquot hearandquot 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 cell PUBLIC HEALTH RELEVANCE Single and limited cell analyses often suffer from cell loss analyte loss non specific binding and sample dilution when performed using liquid transfers between standard consumables We propose to integrate acoustic lysis into a closed system preparative workflow appropriate for low and high throughput applications with special emphasis on LC MS analysis in this project A variety of focused acoustic systems operating at different frequencies and surface modifications to induce cavitation critical to efficient cell lysis will be examined with the optimal choices integrated into a prototype instrument interfaceable to conventional low volume fluid tubing