IDBR: Development of an Optical Sensor for Biological S-Nitrosothiols

最近发现了一种新的细胞通讯机制，S-亚硝基化。这种机制涉及硫原子和一种称为亚硝鎓的化合物之间的化学反应引起的蛋白质变化;这些反应的产物被称为S-亚硝基硫醇。这些化学基团已被证明是重要的翻译后蛋白质修饰，是生物体中许多信号传导途径的一部分。然而，这一领域的研究一直受到缺乏仪器的阻碍，可以测量S-亚硝基硫醇的水平，具有足够的灵敏度和速度，许多情况下感兴趣的生物学家。新的仪器，基于连续波，腔衰荡光谱（CRDS）正在开发的目标是检测一氧化氮（NO）在惰性气体中的单位数万亿分之一的水平。 NO可以通过化学或光化学方式从组织中释放出来，产生的气体被冲入分析仪。 测量光学损耗的仪器是由反射率为99.97%的镜子制成的光学腔，这将导致通过样品的有效吸收路径长度超过3000次。 最近推出的激光器，外腔，量子级联激光器（ec-QCL）将被用来有效地激发腔的光共振，并将在5.2微米附近的NO分子的强拉伸基本跃迁的旋转跃迁。 当激光波长被调谐到NO吸收线时，NO的浓度由腔内光强度衰减速率的增加来确定。基于类似的CRDS仪器在近红外工作的灵敏度，预测比现有技术高约三个数量级的检测下限，这是基于化学发光。 此外，该仪器应提供显着提高的时间分辨率，需要减少样本量，并将区分同位素形式的NO，允许进行标记实验，以确定所释放的NO的化学来源。该项目的第一年将致力于设计，建造和测试的CRDS为基础的仪器。 第二个项目将致力于将CRDS仪器与生物学应用相结合。 除了在生物研究中的应用外，该仪器还可能用于医学诊断。 该项目的进展情况将在http://faculty.virginia.edu/lehmannlab/NO_Detection.html上报告。

A new mechanism for cellular communication, S-nitrosylation, has recently been discovered. This mechanism involves changes in proteins caused by chemical reactions between sulfur atoms and a compound known as nitrosonium; the products of these reactions are known as S-nitrosothiols. These chemical groups have been demonstrated to be important post-translational protein modifications and are part of many signaling pathways in living organisms. However, research in this area has been hampered by the lack of instrumentation that can measure the level of S-nitrosothiols with sufficient sensitivity and speed for many cases of interest to biologists. New instrumentation, based upon continuous wave, cavity ring-down spectroscopy (CRDS) is being developed with a goal to detect Nitric Oxide (NO) in an inert gas at the single digit parts per trillion level. NO can be liberated from tissue either chemically or photochemically and the resulting gas flushed out into analyzer. The instrument measure the optical loss is an optical cavity made from mirrors with 99.97% reflectivity, which will result in an effective absorption pathlength of over 3000 passes through the sample. A recently introduced laser, external cavity, quantum cascade laser (ec-QCL) will be used to efficiently excite the optical resonances of the cavity and will be tuned across the rotational transitions in the strong stretching fundamental transition of the NO molecule near 5.2 micro-meter. The concentration of NO is determined by an increase in the decay rate of intensity of light in the cavity when the laser wavelength is tuned to a NO absorption line. Based upon the established sensitivity of similar CRDS instruments working in the near-InfraRed, a lower detection limit of approximately three orders of magnitude higher than for the existing technology is predicted, which is based upon chemiluminescence. Further, this instrument should give dramatically improved temporal resolution, require reduced sample sizes, and will distinguish isotopic forms of NO, allowing labeling experiments to be done to determine the chemical origin of the released NO. The first year of the project will be devoted towards design, construction, and testing of the CRDS based instrument. The second will be devoted towards interfacing the CRDS instrument with the biological applications. In addition to applications in biological research, the instrumentation could potentially be used for medical diagnostics. Progress on this project will be reported at http://faculty.virginia.edu/lehmannlab/NO_Detection.html.