Development of a Troponin I Biosensor Using a Peptide Obtained through Phage Display

摘要

A small synthetic peptide with nanomolar affinity forncardiac troponin I (TnI), previously identified from anpolyvalent phage displayed library, has been immobilizednon a gold surface for TnI detection. The binding affinitynof gold-immobilized peptides for TnI was studied andncompared with that of phage-immobilized peptides. Quartzncrystal microbalance (QCM), cyclic voltammetry, andnelectrochemical impedance spectroscopy (EIS) were usednto monitor both the immobilization and target bindingnprocesses. All three techniques show that the binding isnspecific for TnI as compared to a streptavidin (SA) control.nThe response curves obtained at TnI concentrationsnranging from 0 to 10 μg/mL, using both QCM and EIS,nwere also compared. For the EIS measurements, thensensitivity was 0.30 ( 0.030 normalized impedance/(μgmL) and the limit of detection (LOD) was 0.34 μg/mL.nUsing the QCM, a sensitivity of 18 ( 1 Hz/(μg/mL) wasnobtained, corresponding to an LOD of 0.11 μg/mL.nAlthough the QCM demonstrated a lower LOD as comparednto EIS, the latter technique exhibited a larger linearndynamic range than QCM. In a relevant tissue culturenmilieu, Minimum Essential Media (MEM), the sensitivitynof the EIS measurement was greater than that obtainednin a phosphate buffer system (PBS). The kinetics of targetnbinding using QCM were analyzed by two independentnmethods, and the dissociation constants (KD ) 66 ( 4nnM and 17 ( 8 nM) were an order of magnitude highernthan that calculated for the polyvalent phage particlesn(KD ) 2.5 ( 0.1 nM). Even though the affinity of thenimmobilized peptides for TnI was somewhat reduced,noverall, these results demonstrate that peptides obtainednfrom the biopanning of phage display librariesncan be readily used as sensing probes in biosensorndevelopment.