(386b) Quartz Crystal Balance (QCM) and Electrochemical Impedance Detection of the Protein Biomarker Troponin I Using Peptides Obtained From the Biopanning of a Phage-Display Library

Immunosensors and immunoassays rely on antibodies and potentially complex detection schemes to monitor binding interactions. We interested in using phage display technology to rapidly identify short unstructured peptides that can bind with high affinity to biomarker proteins. The identified peptides can be easily synthesized and immobilized as recognition probes. We have recently used biopanning to select peptides that bind the cardiac cell stress protein, Troponin I from a polyvalent phage display library. We have now characterized the binding interactions of the free peptides by crystal microbalance (QCM), electrochemical impedance, and cyclic voltammetry. The selectivity and sensitivity of the evolved peptides and the kinetics of Troponin I binding have been measured. All three measurement techniques suggests that the binding between the peptides and the biomarker protein Troponin I still remains when the peptides were immobilized on a gold substrate instead of on the phage particles. The dissociation constant of troponin binding on the QCM was found to be KD = 66 nM by an equilibrium method and KD=17nM by a non-equilibrium method with an on rate of 8.9x103 M-1s-1 and an off rate of 1.5x10-4s-1. These dissociation constants are about an order of magnitude higher than what was obtained when the peptide was immobilized on the phage particles (KD= 2.5nM). The response curves obtained by QCM and by impedance were compared in 0~10 μg/ml range. Impedance spectra showed a limit of detection (LOD) of 0.34μg/ml for Troponin I. There results demonstrate that peptides obtained from phage display experiments can be readily turned into electrochemical biosesnors, and that electrochemical impedance measurements may be the most sensitive electrochemical technique for use in biosensor development.