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-亚硝化。这种机制涉及硫原子与亚硝胺化合物之间的化学反应引起蛋白质的变化；这些反应的产物被称为S亚硝硫醇。这些化学基团已被证明是重要的翻译后蛋白质修饰，是生物体内许多信号通路的一部分。然而，由于缺乏能够对许多生物学家感兴趣的情况下以足够的灵敏度和速度测量S-亚硝硫醇水平的仪器，这一领域的研究一直受到阻碍。基于连续波、腔衰荡光谱(CRDS)的新仪器正在开发中，其目标是在万亿分之一位数的水平上检测惰性气体中的一氧化氮(NO)。NO可以通过化学或光化学方法从组织中释放出来，产生的气体被冲出到分析仪中。测量光学损耗的仪器是由反射率为99.97%的反射镜制成的光学腔体，其有效吸收路径长度将超过3000通过样品。最近引进的外腔量子级联激光器(EC-QCL)将被用来有效地激发腔的光学共振，并将在5.2微米附近的NO分子的强伸缩基态跃迁中通过旋转跃迁进行调谐。当激光波长调谐到NO吸收线时，腔内光强衰减率的增加决定了NO的浓度。根据类似CRDS仪器在近红外下已确立的灵敏度，预测其检测下限将比基于化学发光的现有技术高出约三个数量级。此外，该仪器应显著提高时间分辨率，减少样本大小，并将区分NO的同位素形式，从而能够进行标记实验，以确定释放的NO的化学来源。该项目的第一年将致力于基于CRDS的仪器的设计、建造和测试。第二个项目将致力于将CRDS仪器与生物应用相结合。除了在生物学研究中的应用外，该仪器还可能用于医疗诊断。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.