We propose to develop and demonstrate an AFM-based technology involving a submicron scale probe array, and, by using the array, directly monitor protein interactions from cellular lysates. This technology will allow the analysis of the constituents of cells suspected or known to be involved in oncogenesis. In the technology, AFM tips pick up protein or nucleic acid probes from reservoirs, transfer them to a support, and deposit them at selected array positions. The process is repeated to complete a protein or nucleic acid array, which has submicron space between the array positions (e.g., a 100 x 100 array in about a 0.1 mm x 0.1 mm area). Target proteins are applied to the array, and the binding events of the arrayed molecules are monitored by AFM topography measurements. By carrying out these processes in a miniaturized format and on very small samples, the cost of the analysis will be minimized and the analysis rate will be maximized. Use of a multiple tip array further speeds the analysis rate. The proposed research consists of two phases. In the first phase (R21), we will demonstrate the manipulation of single molecules by the AFM tip and the detection of single molecular binding events by AFM topography measurement. A molecule involved in signal transduction will be selected and monitored by this technique. In the second phase (R33), we will extend the capability of the AFM technique by simultaneously monitoring different molecules associated with parallel signaling events. The immediate purpose of these studies is to determine the relative contributions of established parallel signaling pathways to the transmission of growth signals in cancer of transformed cells. Ultimately, this technique will be used to investigate and determine the assembly of multi-protein complexes isolated from cancer cells.
| Li, Lingyan; Chen, Shengfu; Oh, Seajin et al. (2002) In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy. Anal Chem 74:6017-22 |
