Abstract Cancer is the most threaten disease in this country and deregulations of signal pathways are often responsible for the cancer progression. Many mysteries of cancer related signal pathways have been revealed by current molecular biology research and the detection of protein-protein interactions is one of the critical techniques. The general techniques for protein-protein interactions detection include immuno-precipitation, immuno-fluorescence staining, and fluorescence resonant energy transfer. These techniques can detect specific protein in cultured cell and in tissue and have been applied in most biomedical research. However, all of them have various limitations. For instance, only two molecules could be examined in one experiment and the detection usually require long processing time with complicated procedures and large sample amount. Moreover, the detection of signals currently is still relied on X-ray film exposure or eye-direct observation that may limit the sensitivity and accuracy. We are developing a microchannel detection system in order to avoid these defects and to provide rapid and accurate detection in breast cancer signal transduction research. The principle is that nanochannel fluidics could only allow a single protein complex to pass through the detection point of fluorescence spectroscopy during examination and the result from the single protein complex could provide us most direct information of protein-protein interaction. Since the detection level is down to the single protein complex, it could highly increase the detection sensitivity. Currently, we have successful fabricated different size of microchannels and we recognized that top-down fabrication approach on transparent silicon based wafers could provide the best quality nanochannels. The channel was than integrated to the fluorescence spectroscopy for single fluorescence detection. To test the performance of the device, fluorescence antibodies were loaded into the channel and we successfully detected a single fluorescence signal during the experiment. Subsequently, we detect demonstrated that epidermal growth factor receptor (EGFR) interact with its downstream proteins Src and STAT3 in a single complex after epidermal growth factor (EGF) stimulation. However, only 1.8% of EGFR interact with both Src and STAT3 at the same time which suggests that EGFR/Src/STAT3 is a transition product in the dynamic of EGFR signal transduction. Thus, our system provides the interaction information from a single protein complex with the ratio of interactive proteins to dissect the signal transduction in dynamic aspect and will offer an effective technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4017.