Coiled-coil peptide based sensor for ultra-sensitive thrombin detection

• New biosensor platform based on coiled-coil peptide modified comb structured gold microelectrode array has been fabricated.
• Use of CCP with thrombin cleavage site provides the opportunity to detect thrombin linearly from 10 fg/ml to 1pg/ml concentration range with in 10 min.
• Platform helps to understand the catalytic activity mechanism of thrombin.

Comb structured gold microelectrode array (CSGMA) functionalized with self-assembled monolayer of thiol terminated coiled-coil peptide (CCP) linked together by the thrombin specific cleavage site (Leu–Val–Pro–Arg–Gly–Ser) has been used to fabricate an ultrasensitive, disposable, electrochemical thrombin biosensor. CCP with thiol at one end provides the ease of CSGMA functionalization and the presence of thrombin specific peptide in the middle of coiled-coil peptide provides site for thrombin capture and detection. CCP/CSGMA electrodes were characterized using label-free electrochemical impedance (EIS) technique and exposed to solutions with different thrombin concentrations for its estimation. Results reveal that CCP/CSGMA electrodes have a limit of detection (LOD) of 10 fg/ml (28 fM) and are able to detect catalytic activity of thrombin within 30 min time frame. CCP/CSGMA electrodes were found to be selective against other IgG anti-bodies such as DO1 and HA. Thus, CCP/CSGMA electrodes provide high specificity toward thrombin detection and mechanistic details of binding and cleavage process.