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

机译:使用通过噬菌体展示获得的肽来开发肌钙蛋白I生物传感器

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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 (mu)g/mL, using both QCM and EIS, were also compared. For the EIS measurements, the sensitivity was 0.30 +- 0.030 normalized impedance/((mu)g/mL) and the limit of detection (LOD) was 0.34 (mu)g/mL. Using the QCM, a sensitivity of 18 +- 1 Hz/((mu)g/mL) was obtained, corresponding to an LOD of 0.11 (mu)g/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) velence 66 +- 4 nM and 17 +- 8 nM) were an order of magnitude higher than that calculated for the polyvalent phage particles (K_(D) velence 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.
机译:先前已从多价噬菌体展示文库中鉴定出的对心脏肌钙蛋白I(TnI)具有纳摩尔摩尔亲和力的小型合成肽已固定在金表面上,用于TnI检测。研究了金固定肽对TnI的结合亲和力,并与噬菌体固定肽进行了比较。石英晶体微量天平(QCM),循环伏安法和电化学阻抗谱(EIS)用于监测固定化和靶结合过程。与抗生蛋白链菌素(SA)对照相比,所有三种技术均显示该结合对TnI具有特异性。还比较了同时使用QCM和EIS在TnI浓度为0至10μg/ mL时获得的响应曲线。对于EIS测量,灵敏度为0.30±0.030归一化阻抗/(μg/ mL),并且检测极限(LOD)为0.34μg/ mL。使用QCM,灵敏度为18 +/- 1 Hz /(μg/ mL),对应于LOD为0.11μg/ mL。尽管与EIS相比,QCM的LOD较低,但后一种技术比QCM的线性动态范围更大。在相关的组织培养环境中,最低必需培养基(MEM),EIS测量的灵敏度高于在磷酸盐缓冲液系统(PBS)中获得的灵敏度。通过两种独立方法分析了使用QCM进行靶标结合的动力学,解离常数(K_(D)velence 66 + -4 nM和17 +-8 nM)比多价噬菌体颗粒计算的解离常数高一个数量级。 (K_(D)速度2.5±0.1 nM)。总体而言,尽管固定肽对TnI的亲和力有所降低,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。

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