Development of a troponin I biosensor using a peptide obtained through phage display.
A small synthetic peptide with nanomolar affinity for cardiac troponin I (TnI), previously identified from a polyvalent phage displayed library, has been immobilized on a gold surface for TnI detection. The binding affinity of gold-immobilized peptides for TnI was studied and compared with that of phage-immobilized peptides. Quartz crystal microbalance (QCM), cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were used to monitor both the immobilization and target binding processes. All three techniques show that the binding is specific for TnI as compared to a streptavidin (SA) control. The response curves obtained at TnI concentrations ranging from 0 to 10 μg/mL, using both QCM and EIS, were also compared. For the EIS measurements, the sensitivity was 0.30 ± 0.030 normalized impedance/(μg/mL) and the limit of detection (LOD) was 0.34 μg/mL. Using the QCM, a sensitivity of 18 ± 1 Hz/(μg/mL) was obtained, corresponding to an LOD of 0.11 μg/mL. Although the QCM demonstrated a lower LOD as compared to EIS, the latter technique exhibited a larger linear dynamic range than QCM. In a relevant tissue culture milieu, Minimum Essential Media (MEM), the sensitivity of the EIS measurement was greater than that obtained in a phosphate buffer system (PBS). The kinetics of target binding using QCM were analyzed by two independent methods, and the dissociation constants (K(D) = 66 ± 4 nM and 17 ± 8 nM) were an order of magnitude higher than that calculated for the polyvalent phage particles (K(D) = 2.5 ± 0.1 nM). Even though the affinity of the immobilized peptides for TnI was somewhat reduced, overall, these results demonstrate that peptides obtained from the biopanning of phage display libraries can be readily used as sensing probes in biosensor development.
Wu
Jun
J
Department of Chemical Engineering, Columbia University, New York, New York, USA.
Cropek
Donald M
DM
West
Alan C
AC
Banta
Scott
S
D015374:Biosensing Techniques / Q000379:methods*; D058266:Dielectric Spectroscopy / Q000379:methods; D055664:Electrochemical Techniques / Q000379:methods; D006046:Gold / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry; D007700:Kinetics; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods; D019210:Troponin I / Q000032:analysis*
D055767:Immobilized Proteins; D019151:Peptide Library; D010455:Peptides; D019210:Troponin I; D006046:Gold