| Summary: | 碩士 === 國立中正大學 === 化學工程所 === 94 === Guanosine 3,5-cyclic monophosphate (cGMP) produced from guanosine triphosphate (GTP) via soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC) is an important secondary messenger in cells. Commonly used methods for measurement of cGMP are the competitive enzyme immunoassay (EIA) and radioimmunoassay. However, the protocol for EIA is tedious and the radioimmunoassay is unsafe for personnel. To overcome the above disadvantages, we developed an optical fiber modified with a self-assembled monolayer of nano-gold particles conjugated with anti-cGMP antibody to determine cGMP concentration based on localized surface plasmon resonance (LSPR). The optimal antibody dilution and incubation time for preparing the probe are 1:250 and 2 h, respectively. Particularly, this senor shows much better sensitivity than EIA with a cGMP detection range of 2.5×10-3-102 pM. After measurement, the cGMP bound to the probe can be washed out using DPBS (pH 6) containing 0.05% Tween 20 and the probe can be reused once for the unknown sample with cGMP concentration less than 20 pM. In addition, the prepared probe can be stored at 4˚C up to 1 week without losing significant sensitivity. Because acetylated cGMP has higher affinity with cGMP antibody than cGMP, the sensitivity of measuring acetylated cGMP by the probe is increased by 3 orders of magnitude as compared to the measurement of cGMP. Finally, the reliability of the probe was confirmed by measuring the cGMP concentration in the cell extract of human aortic smooth muscle cells under various treatments using a commercial cGMP EIA kit.
|
|---|
