Protein functionalised self assembled monolayer based biosensor for colon cancer detection

We report results of the studies relating to the fabrication of a surface plasmon resonance (SPR) based label-free immunosensor for real-time monitoring of endothelin-1 (ET-1), a colon cancer biomarker. A gold disk modified with a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) was functionalised via covalent immobilization of monoclonal anti-ET-1 antibodies using EDC-NHS (1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride, N-hydroxy succinimide) chemistry. This immunosensing platform (ethanolamine/anti-ET-1/11-MUA/Au) was characterized via atomic force microscopy (AFM), contact angle (CA) and Fourier transform infrared (FT-IR) spectroscopic techniques. The fabricated SPR electrode was further used to detect ET-1 in the broad concentration range 2-100 pg mL^-1, with a detection limit of 0.30 pg mL^-1 and remarkable sensitivity of 2.18 m^o pg^-1mL. The adsorption mechanism was studied using monophasic model and the values of association (k_a) and dissociation (k_d) constants for anti-ET-1 and ET-1 binding were calculated to be 4.4 ± 0.4 × 10⁵ M^-1 s^-1 and 2.04 ± 0.0003 × 10^-3 s^-1, respectively. The results obtained via analysis of serum samples of colorectal cancer patients were found to be in good agreement with those obtained from enzyme-linked immunosorbent assay (ELISA) technique. Further, electrochemical studies were performed to prove the efficacy of the fabricated platform as a point of care device for the detection of ET-1.