Surface infrared spectroscopic studies on cellular dynamic processes and the application to cell chips

在本研究中，我们开发了基于多重内反射红外光谱（MIR-IRAS）的细胞动力学原位实时监测方法。我们的研究项目主要集中在（1）发展原位监测细胞生长的IRAS方法，（2）发展非标记IRAS检测细胞死亡及其在细胞毒检测中的应用。此外，我们还研究了生物分子相互作用的非标记检测，例如（3）抗原-抗体相互作用和（4）溶液相和硅表面的DNA杂交。 为了开发实时监测细胞动力学的方法，我们首先在FTIR样品室中构建细胞培养条件。放置在FTIR样品室中的细胞室中的温度和湿度分别控制为37 °C和> 80%。由于样品室需要用不含水和二氧化碳的空气吹扫，因此通过硅胶管将含CO_2的加湿混合气体引入室中。利用该装置，我们证实了HL-60细胞在培养箱中孵育24小时后的细胞活力和增殖。然后用MIR-IRAS原位监测Tween 20的细胞毒性作用。结果表明，可以通过分析酰胺II峰强度原位监测细胞死亡。此外，我们成功地在实时监测的动态过程中诱导凋亡，通过使用MIR-IRAS。我们还研究了抗原-抗体相互作用和DNA杂交的无标记检测，并证明了MIR-IRAS通过仔细分析光谱特征对区分特异性和非特异性吸附特别有用。因此，我们的方法可以应用于各种细胞事件的原位监测，以及个人的生物分子相互作用作为高性能细胞芯片和生物芯片的非标记检测方法。

In this research, we have developed m situ real-time monitoring method for cell dynamics on the basis of infrared spectroscopy with the multiple internal reflections (MIR-IRAS). Our research projects were focused on (1) development of IRAS method for in situ monitoring of cell growth, (2) development of non-label IRAS detection of cell death and its application to cytotoxic assay. In addition, we also examined non-label detection of biomolecular interactions, such as (3) antigen-antibody interactions and (4) DNA hybridization both in solution phase and at the silicon surfaces. For the development of the real-time monitoring of cell dynamics, we started with construction of cell culture condition in a FTIR sample room. The temperature and humidity in a cell chamber placed in the FTIR sample room was controlled to be 37 °C and >80%, respectively. Since the sample room needs to be purged with air which was free from water and carbon dioxide vapor, a humidified gas mixture containing CO_2 was introduced to the chamber through silicone tubes. By using this apparatus, we have confirmed the cell viability and proliferation of HL-60 cells after 24-h incubation in the chambers. Then the cytotoxic effect by Tween20 was in-situ monitored with MIR-IRAS. It was demonstrated that the cell death can be monitored in-situ by analyzing the amide II peak intensity. In addition, we succeeded in real-time monitoring of the dynamic processes upon apoptosis induction by using MIR-IRAS. We also examined label-free detection of antigen-antibody interactions and DNA hybridization and demonstrated that MIR-IRAS is particularly useful for discriminating specific and non-specific adsorption by careful analysis of spectral features. Thus our method can be applied to in-situ monitoring of various cellular events as well as individual biomolecular interactions as non-label detection method for high-performance cell-chips and bio-chips.