High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, 230031, China.
The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Center for Bioanalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui, 230026, China.
Chem Commun (Camb). 2022 Jul 28;58(61):8528-8531. doi: 10.1039/d2cc02361j.
Caspase-3 plays a vital role in cell apoptosis and related diseases. The detection and characterization of endogenous active caspase-3 are of immense value not only for mechanism studies of apoptosis but also for the diagnosis and treatment of apoptosis-related diseases. Here, an electron paramagnetic resonance (EPR)-based enzymatic assay was developed for the detection of caspase-3 activity both and in apoptosis cells. This assay uses a sandwich-like probe composed of a caspase-3-specific peptide segment (DEVD) conjugated to an EPR-detectable nitroxide spin label and magnetic beads (MBs). Cleavage of the "Nitroxide-Peptide-MBs" sandwich probe caspase-3 will release the nitroxide, which is readily detected by EPR after magnetic separation, resulting in a distinct EPR "off/on" transition. This assay takes advantage of the specific cleavage of DEVD-containing peptides by caspase-3 for high specificity, magnetic beads for fast magnetic separation, and EPR spectroscopy for considerably high detection sensitivity (LODs for caspase-3 are 116 nM at 60 min and 58 nM at 120 min). Importantly, the assay was proven to be compatible with complex biological samples and can detect the endogenous active caspase-3, thereby providing potential applications in the screening of protease-targeted drugs and the diagnosis of protease-associated diseases.
Caspase-3 在细胞凋亡及相关疾病中起着至关重要的作用。内源性活性 caspase-3 的检测和鉴定不仅对凋亡机制的研究具有重要意义，而且对凋亡相关疾病的诊断和治疗也具有重要意义。本研究开发了一种基于电子顺磁共振（EPR）的酶联免疫吸附测定法，用于检测细胞凋亡过程中和细胞内的 caspase-3 活性。该检测方法使用一种夹心样探针，由与 EPR 可检测的氮氧自由基自旋标记物和磁珠（MBs）偶联的 caspase-3 特异性肽段（DEVD）组成。"Nitroxide-Peptide-MBs" 夹心探针被 caspase-3 切割后，会释放出氮氧自由基，经磁分离后，EPR 可轻松检测到该自由基，从而产生明显的 EPR“开/关”跃迁。该检测方法利用 caspase-3 对含有 DEVD 的肽段的特异性切割，具有高特异性、磁珠的快速磁分离以及 EPR 光谱法的高检测灵敏度（在 60 分钟和 120 分钟时，caspase-3 的检测限分别为 116 nM 和 58 nM）。重要的是，该检测方法与复杂的生物样本兼容，并且可以检测内源性活性 caspase-3，从而为蛋白酶靶向药物的筛选和蛋白酶相关疾病的诊断提供了潜在的应用。
