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)开发用于原位监测细胞生长的IRAS方法，(2)开发用于细胞死亡的非标记IRAS检测及其在细胞毒性测定中的应用。此外，我们还研究了生物分子相互作用的非标记检测，如(3)抗原-抗体相互作用和(4)溶液和硅表面的DNA杂交。为了实现细胞动力学的实时监测，我们从构建FTIR样品室的细胞培养条件开始。放置在FTIR样品室的细胞室的温度和湿度分别控制在37°C和bbb80 %。由于样品室需要用不含水和二氧化碳蒸汽的空气进行净化，因此通过硅胶管将含有CO_2的加湿气体混合物引入样品室。通过该装置，我们证实了HL-60细胞在培养室中培养24小时后的细胞活力和增殖能力。然后用MIR-IRAS原位监测Tween20的细胞毒作用。结果表明，通过分析酰胺II峰强度，可以实时监测细胞的死亡情况。此外，我们利用MIR-IRAS成功地实时监测了细胞凋亡诱导的动态过程。我们还研究了抗原-抗体相互作用和DNA杂交的无标记检测，并证明通过仔细分析光谱特征，MIR-IRAS对于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以及个体生物分子相互作用的原位监测，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.