Multiplexing determination of lung cancer biomarkers using electrochemical and surface-enhanced Raman spectroscopic techniques

A simple multiplexed electrochemical and surface-enhanced Raman spectroscopic (SERS) immunosensor is developed for simultaneous detection of carcinoembryonic antigen (CEA) and cytokeratin-19 (CK-19). We find a kind of Raman dye (thionine and Nile blue A)-decorated resin microspheres by pi-pi stacking interaction, which show both strong SERS signals and electrochemical redox characteristic peaks. Aminosalicylic acid-based resin (AAR) microspheres are synthesized by a template-free method for the first time. The Au nanoparticle (AuNP) coated AAR microspheres (AuNPs/AAR) are prepared by a convenient reduction approach. A sandwich structure contains Raman dye-labeled AuNPs/AAR with the first antibody, the second antibody immobilized on the electrode modified chitosan stabilized AuNPs, and target antigens. Thus, in the presence of the target antigens, the Raman dye-labeled AuNPs/AAR could be bonded to the modified electrode surface by antibody-antigen-antibody interactions. Compared with thionine, Nile blue A has different Raman signals and electrochemical characteristic peaks. As a result, a simultaneous immunoassay for CEA and CK-19 based on multiple labels is developed by using electrochemical and SERS immunoassays. The prepared immunoassay for detection of CEA and CK-19 shows high sensitivity, selectivity, low detection limit and long term stability. The lowest detectable concentration is 0.01 ng mL(-1) and 0.04 ng mL(-1) for CEA and CK-19 at a signal to noise rate of 3, respectively. The proposed immunosensor can be applied to determine CEA and CK-19 in human blood serum with favorable results. In addition, the electrochemical and SERS immunosensor has potential application in the diagnosis and treatment of lung cancer in the field of clinical research.