Biomarkers of clinical activity are critical for targeted anti-cancer therapy development and are becoming important for the care of individual patients. In a prototypical tyrosine kinase pathway, governed by the epidermal growth factor receptor (EGFR), functional activity of the pathway is assessed by the phosphorylation status of EGFR and downstream signaling intermediaries such as phospho-ERK and phospho-Akt. Interventions that block the function of EGFR (such as tyrosine kinase inhibitors or monoclonal antibodies) may lead to lack of phosphorylation of these downstream intermediaries. Traditionally, phosphorylated proteins have been analyzed by Western blots performed on tumor protein extracts from as many as 106 cells. A method is proposed that can utilize small sample to achieve a similar level of detection of phopshoproteins in the EGFR pathway. Tumor cells would be isolated from peripheral blood obtained before and after administration of an EGFR targeted therapy and the phosphorylation status of key EGFR pathway intermediates would be analyzed. The proposed approach to sample preparation is based on two molecular recognition events: capture of analytes of interest (in this case, tumor cells, or their components after lysis) on non-magnetic beads carrying receptors as well as "codes";and then binding these beads by magnetic beads carrying "anticodes". The codes and anti-codes can simply be two complementary DNA strands. Unlike single-step magnetic-bead capture, the proposed method allows simultaneous capture of multiple analytes by incubation with different bead types at the same time. They are then sorted by consecutive exposure to various types of "decoding" beads. After processing the samples to simultaneously capture multiple analytes of interest, the sample will be loaded onto electrowetting (EW) biochip. The sample will be subdivided into droplets of similar size and run past droplets containing decoding magnetic beads. The ability of EW chip to rapidly process multiple droplets enables the sorting procedure. For example, different aliquots of bead suspension can be reacted with the batches of magnetic beads in different sequences, to avoid bias due to non-specific binding. The ultimate advantage is sample concentration by at least 103x and removal of background material. The sample need not be subdivided which increases the sensitivity and speed of multiplexed assays while allowing minimally-invasive sample collection. Moreover, the final analysis - immunoassay, or PCR or RTPCR, - can be performed on same chip, taking advantage of the ultimate sensitivity and dynamic range of these liquid-phase assays. Advanced Liquid Logic, Inc. will team with collaborators at Duke University's Comprehensive Cancer Center to execute this project.