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

细胞动态过程的表面红外光谱研究及其在细胞芯片中的应用

基本信息

批准号：
17206004
负责人：
NIWANO Michio
金额：
$ 32.03万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17206004/
关键词：
Cell chip Infrared spectroscopy Surface Biochip Semiconductor 細胞 DNA ハイブリダイゼーション

项目摘要

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.

在本研究中，我们发展了基于多次内反射红外光谱(MIR-IRAS)的细胞动力学实时监测方法。我们的研究项目主要集中在：(1)建立原位监测细胞生长的IRAS方法；(2)发展检测细胞死亡的非标记IRAS方法，并将其应用于细胞毒检测。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)溶液中和硅表面的DNA杂交。为了发展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入手。放置在FTIR样品室的电池室内的温度和湿度分别控制在37℃和&gt；80%。由于样品室需要用不含水和二氧化碳蒸气的空气净化，因此通过硅胶管将含有二氧化碳的湿化气体混合物引入到样品室。利用该装置，我们证实了HL-60细胞在小室中孵育24小时后的细胞活力和增殖能力。用MIR-IRAS原位检测吐温20的细胞毒作用。结果表明，通过对酰胺II峰强度的分析，可以对细胞死亡进行原位监测。此外，我们还利用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-抗体相互作用的无标记检测和DNA杂交，并通过仔细分析光谱特征证明了MIR-IRAS在区分特定和非特定吸附方面特别有用。因此，我们的方法可以作为高性能细胞芯片和生物芯片的非标记检测方法，应用于各种细胞事件的现场监测以及单个生物分子的相互作用。