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

Abstract

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.

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

“…The frequency change on immobilization to the gold modified QCM crystal corresponds to a mass loading as comparable to what has been observed in previous work, as computed by the Sauerbrey equation. 21 On addition of 10 mM CaCl 2 , a change in both frequency (12 Hz) and resistance (À1 ohm) was observed [ Fig. 1(b)].…”

“…According to our previous work, using a manufacturer provided relationship, a 1-ohm resistance change contributes about 2 Hz to the total frequency change. 21 Therefore, most of the frequency change ($10 Hz) is because of loss of mass at the surface, suggesting that a dehydration process likely takes place on beta roll formation. After switching to calcium-free buffer, a return to baseline was observed.…”

“…15 QCM experiments were performed as described previously. 21 All experiments were carried out in Tris buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Calcium chloride and magnesium chloride were used as the calcium and magnesium ion sources, respectively, and added as indicated.…”

“…The quartz crystal microbalance (QCM) is a sensitive and versatile tool that has been applied to detect biomolecular interactions and conformational changes in peptides and proteins. 20,21 For example, the calcium-induced conformational change of calmodulin has been studied by QCM, where it was shown that the dehydration process and increased rigidity of the protein on calcium binding was detectable as an increase in frequency. 22 Considering the significant conformational change of the RTX domain in response to calcium, we hypothesized that this change would be detectable by QCM and that its behavior would depend on the N-terminal or C-terminal orientation of the peptide due to previous experiments where such dependencies have been observed.…”

Biomimetic Sensors for Rapid Testing of Water Resources

“…The quantitative response achieved for both QCM and EIS validates that both methods could be used as a sensing technique in quantitative ALT measurements. In addition to the generation of this ALT-specific recognition element, we have developed a recognition element for the cardiac specific biomarker, troponin I (TnI) [13,14]. Again, phage display was employed to generate the recognition peptide while QCM and EIS validated and quantified the binding events and specificity of the peptide for the target, TnI.…”