Department of Chemical Engineering, Columbia University, New York, New York, USA.
Anal Chem. 2010 Oct 1;82(19):8235-43. doi: 10.1021/ac101657h.
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.
一种对心肌肌钙蛋白 I（TnI）具有纳摩尔亲和力的小合成肽，先前从多价噬菌体展示文库中鉴定出来，已被固定在金表面上用于 TnI 检测。研究了金固定肽对 TnI 的结合亲和力，并将其与噬菌体固定肽的结合亲和力进行了比较。使用石英晶体微天平（QCM）、循环伏安法和电化学阻抗谱（EIS）监测固定化和靶标结合过程。所有三种技术都表明，与链霉亲和素（SA）对照物相比，该结合是特异性的。使用 QCM 和 EIS ，在 TnI 浓度范围为 0 至 10 μg/mL 的情况下获得的响应曲线也进行了比较。对于 EIS 测量，灵敏度为 0.30 ± 0.030 归一化阻抗/（μg/mL），检测限（LOD）为 0.34 μg/mL。使用 QCM ，获得了 18 ± 1 Hz/（μg/mL）的灵敏度，对应于 0.11 μg/mL 的 LOD。尽管 QCM 的 LOD 低于 EIS，但后者技术的线性动态范围大于 QCM。在相关的组织培养环境中，最小必需培养基（MEM）中，EIS 测量的灵敏度大于在磷酸盐缓冲系统（PBS）中获得的灵敏度。使用 QCM 分析了靶标结合的动力学，两种独立的方法都分析了动力学，解离常数（K(D) = 66 ± 4 nM 和 17 ± 8 nM）比从多价噬菌体颗粒计算出的解离常数（K(D) = 2.5 ± 0.1 nM）高一个数量级。尽管固定化肽对 TnI 的亲和力有些降低，但总的来说，这些结果表明，从噬菌体展示文库的生物淘选获得的肽可以很容易地用作生物传感器开发中的传感探针。
