Bioluminescent RAPPID Sensors for the Single-Step Detection of Soluble Axl and Multiplex Analysis of Cell Surface Cancer Biomarkers.

Abstract

Early diagnosis of cancer is essential for the efficacy of treatment. Our group recently developed RAPPID, a bioluminescent immunoassay platform capable of measuring a wide panel of biomarkers directly in solution. Here, we developed and systematically screened different RAPPID sensors for sensitive detection of the soluble fraction of Axl (sAxl), a cell surface receptor that is overexpressed in several types of cancer. The best-performing RAPPID sensor, with a limit of detection of 8 pM and a >9-fold maximal change in emission ratio, was applied to measure Axl in three different contexts: clinically relevant sAxl levels (∼0.5 and ∼1 nM) in diluted blood plasma, proteolytically cleaved Axl in the cell culture medium of A431 and HeLa cancer cells, and Axl on the membrane of A431 cells. We further extended the sensor toolbox by developing dual-color RAPPID for simultaneous detection of Axl and EGFR on A431 and HeLa cells, as well as an AND-gate RAPPID that measures the concurrent presence of these two cell surface receptors on the same cell. These new RAPPID sensors provide attractive alternatives for more laborious protein detection and quantification methods such as FACS and immunostainings, due to their simple practical implantation and low intrinsic background signal.