We will develop novel imaging technologies for real-time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g. DNA region, RNA region, protein, small molecule), and to do so at relatively low cost. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short-time to facilitate practical applications. The general scheme is based on novel concepts for single- cell and single-molecule detection and characterization recently demonstrated in our laboratory. Four distinct but interrelated goals are identified: (1) development of a single molecule detection system which permits rapid and highly confident detection of hybridization to a DNA probe of binding to an antibody in the presence of a large excess of unhybridized probe molecules of free antibodies, respectively. This will lead to single-event homogenous assays of molecular alterations of DNA, proteins, or small molecules in biological tissues; (2) development of high-speed high-throughput specialized data treatment software for rapid """"""""on-line"""""""" analysis of the images with an emphasis on confidence in target recognition and reduction in post-imaging data work-up; (3) development of a microscale single-cell manipulation and reaction system for the rapid detection and identification of specific target cells in the presence of a large excess of other cells. It may then be possible to detect directly targeted species in biological tissues without introducing additional probes; and (4) demonstration of actual analysis of human cells and tissue samples, with outside collaboration, to evaluate and to validate the technology developed, and to further optimize the performance regarding speed, ruggedness and accuracy. The first two goals will be the focus of the R21 Phase and the last two goals will be the focus of the R33 Phase of this application.
| Li, Jiangwei; Lee, Ji-Young; Yeung, Edward S (2006) Quantitative screening of single copies of human papilloma viral DNA without amplification. Anal Chem 78:6490-6 |
| Isailovic, Dragan; Sultana, Ishrat; Phillips, Gregory J et al. (2006) Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits. Anal Biochem 358:38-50 |
