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.
We have developed a new instrument that can detect trace levels of Nitric Oxide (NO) gas in other gases. The instrument uses a technique known as continuous wave cavity ring-down spectroscopy, which the PI pioneered and is now widely used in research and in industrial applications where ultrahigh sensitivity is required. The present project was the first time the PI has extended his work on this technique into the mid-IR spectral region. The project realized a sensitivity to detect NO gas down to levels of less than 50 parts per trillion, far surpassing the sensitivity of the currently widely used detection instruments for NO that detect chemiluminescence of NO gas when it reacts to produce nitrogen dioxide gas. The present approach is superior in other ways as well, including allowing absolute concentration to be determined without the need of calibration samples, which are very hard to produce accurately for a reactive chemical species such as NO. Also, we can discriminate different isotopic forms of NO, which is useful in many applications. The instrument was developed to aid in Biological research where the ability to detect trace NO concentrations with sufficient sensitivity and speed have hampered research into the role of NO attached to proteins which is a mechanism of signaling and post translational control in biology. The new technology will likely also find medical applications as well since it can likely be adopted to monitor NO levels in human breath, which is a useful diagnostic tool for a number of human health conditions, including Asthma and Cystic Fibrosis. Below is a schematic of the instrument.
