Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
ACS Nano. 2014 May 27;8(5):4883-92. doi: 10.1021/nn500840x. Epub 2014 Apr 18.
Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.
细胞凋亡是一种在胚胎发生、衰老和各种疾病中发挥重要作用的生物学过程。在凋亡过程中，细胞经历一系列形态和分子事件，如起泡、细胞收缩、蛋白水解和核 DNA 片段化。在分子水平上研究这些事件对于更全面地了解凋亡的复杂机制至关重要；然而，在单个活细胞尺度上实时直接观察形态和分子事件仍然是一个挑战。在此，我们利用之前描述的等离子体增强瑞利/拉曼光谱技术，在凋亡诱导剂处理后实时直接监测细胞凋亡过程中的形态和分子事件。对细胞核周围 DNA/蛋白质组成的光谱分析揭示了三种凋亡分子事件的发生和动态：蛋白质变性、蛋白水解和 DNA 片段化。分子事件的动力学用于创建单细胞中凋亡事件的时间分布。结果表明，过氧化氢诱导的凋亡过程中发生的事件顺序是通过二硫键断裂导致蛋白质变性以及 DNA 片段化，随后是疏水性氨基酸暴露导致蛋白质解开，最后是蛋白质降解。这些结果表明，使用这种时间依赖性等离子体增强振动成像技术研究不同试剂（如抗癌药物）诱导的其他凋亡分子途径的详细机制具有潜力。结论中指出，生物分子在溶液中的 SERS 光谱与被聚集纳米颗粒的等离子体场增强的活细胞中观察到的光谱之间存在预期的很大差异。
