Basic Research Laboratory, Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Republic of Korea.
Department of Chemistry, Institute of Interdisciplinary Convergence Research, Research Institute of Chem-Bio Diagnostic Technology, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea.
Bioelectrochemistry. 2022 Jun;145:108090. doi: 10.1016/j.bioelechem.2022.108090. Epub 2022 Feb 24.
Caspase-3, a cysteine-dependent protease, is considered a reliable molecular biomarker for the diagnosis and prognosis of apoptosis-related diseases. In this study, we demonstrated a phage-based electrochemical biosensor for the evaluation of cell apoptosis by the sensitive and specific detection of caspase-3. Specifically, for screening of affinity peptide-displayed phages, phage display was performed using M13 phage libraries (cyclic forms of peptides), and we identified potential affinity peptide-displayed phage clones with the sequence CPTTMWRYC. After characterization of its binding affinity using enzyme-linked immunosorbent assay, whole phage particles were covalently attached to a gold surface using coupling chemistry (MUA-EDC/NHS). The developed phage sensor was characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), electrochemical analysis using cyclic voltammetry (CV), and square wave voltammetry (SWV). Under optimal conditions, the affinity peptide-displayed phage sensor showed a good binding affinity (K = 0.13 ± 0.56 μM) and limit of detection (0.39 μM) for caspase-3 detection. Furthermore, developed phage sensor could be monitored the response of apoptotic HeLa cells by detecting caspase-3 activity. This work should stimulate the development of efficient alternative caspase-3 detection methods for the diagnosis and prognosis of apoptosis-related diseases.
半胱氨酸依赖性蛋白酶 caspase-3 被认为是诊断和预测与细胞凋亡相关疾病的可靠分子生物标志物。在这项研究中，我们展示了一种基于噬菌体的电化学生物传感器，通过灵敏和特异性检测 caspase-3 来评估细胞凋亡。具体来说，为了筛选亲和肽展示噬菌体，我们使用 M13 噬菌体文库（肽的环状形式）进行噬菌体展示，并鉴定出具有序列 CPTTMWRYC 的潜在亲和肽展示噬菌体克隆。在用酶联免疫吸附试验（ELISA）对其结合亲和力进行表征后，我们使用偶联化学（MUA-EDC/NHS）将完整的噬菌体颗粒共价连接到金表面。通过 X 射线光电子能谱（XPS）、原子力显微镜（AFM）、扫描电子显微镜（SEM）、循环伏安法（CV）和方波伏安法（SWV）对开发的噬菌体传感器进行了表征。在最佳条件下，亲和肽展示噬菌体传感器对 caspase-3 检测表现出良好的结合亲和力（K = 0.13 ± 0.56 μM）和检测限（0.39 μM）。此外，开发的噬菌体传感器可以通过检测 caspase-3 活性来监测凋亡 HeLa 细胞的反应。这项工作应该会激发人们开发用于诊断和预测与细胞凋亡相关疾病的高效替代 caspase-3 检测方法。
