The Study On An Optical Nitric Oxide Biosensor And Its Application To The Research Into Releasing Of NO Donor In Vitra And The Effect Of Drugs On Macrophage Producing Nitric Oxide

Nitric oxide (NO) is an important molecule of messenger and effect, and plays an important role in physiological and pathological processes. Since 1990, many scientists have focused on the studying of NO. NO is a simple gaseous compound and its lifetime is very short (t_1/2<6s) in presence of oxygen. Therefore analytical methods for NO gain much attention for its difficult detecting. At present, most of detection methods for NO are indirect which detect the metabolic products of NO such as NO₂^- or (and) NO₃^-. Griess colorimetry is one of these methods.A NO biosensor was built with using CytC as the sensitive material. When NO reacts with iron porphyrin, the absorption spectrum of CytC changes at special wavelength which is related to the concentration of NO. The assay method of NO was developed by the NO biosensor with the acidified NaNO₂ solution as NO source. And the releasing of NO donor in vitra and the effect of drugs on macrophage producing nitric oxide were also investigated.The whole study includes several parts as follow: 1. Development of optical NO biosensor based on absorptionA NO biosensor based on absorption was built by immobilized CytC with several different methods, including gold colloid self-assemble film, chitosan covalent link film and sol-gel encapsulation film. Overall validation of these films was carried out by comparing their apparent characters and response properties to NO. Al₂O₃/SiO₂ complex film was determined for its short response time, highsensitive and less interference. The standard curve was linear within the range of 40.0-360.0umol/L of NO in 3><10"2mol/LHCl solution (r=0.9988), the limit of detection was 6.0|j.mol/L, with-day precision was 1.3%, day-to-day precision was 3.2%, the recoveries of low, middle and high concentration were 100.14%, 103.17% and 96.64%.2. The study of releasing of NO donor in vitra by NO biosensorThe passageway and the mechanism of sodium nitroprusside (SNP), a NO donor, releasing NO was investigated by NO biosensor. The detection of NO released by SNP was carried out in different physiological solutions, which take place in three different mechanisms. The result revealed that photolysis and thiol reducing causes SNP releasing NO. The standard curve was linear within the range of 40.0-360.0umol/L of NO in acidified physiological solution(r=0.9992), the limit of detection was 4.0umol/L, with-day precision was 2.3%, day-to-day precision was 3.4%, the recoveries of low, middle and high concentrate were 94.25%, 96.11% and 99.38%. Both L-Cys and L-GSH have influence on CyC film in neutral solution and have no influence in acidic solution. NO in solution was measured by biosensor and Griess colorimetry respectively, and no significant difference between results of the two methods was found. The biosensor system provides a good method for the study on NO donor releasing NO in vitra.3. Investigation of NO produced with macrophage stimulated by drugsThe detection method of NO produced by macrophage was built. The standard curve was linear within the range of 20.0-180.0nmol/L of NO in acidified culture solution(r=0.9959), the limit of detection was 5.0umol/L, with-day precision was 3.5%, day-to-day precision was 7.9%. The culture solution and drugs (sulfated polysaccharides, Lentinan. Mannatide) did not interfere CytC films. The detection of NO in culture solution was carried out by using interferon—y (IFN-y) and lipopolysaccharide (LPS) as positive control trial. The concentration of NO is below the detection limit of biosensor, but there was no significant difference between results of biosensor and Griess colorimetry. So the biosensor can be alsoused as a semi-quantitative analysis method and be used to screening drugs preliminarily.