Oxygen fluorescence detection

Kurosawa S, Yoshimura T, Kurosawa H, Chiba K. 1995. Simultaneous determination of 18-oxygenated corticosteroids by high performance liquid chromatography with fluorescence detection. J Liq Chromatogr 18 2383-2396. 

Hanson, K. M., and Clegg, R. M. 2005. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol. Biol. 289 413-22. 

The mobile phase plays an important part in the fluorescence of a molecule. Unless chosen with care, the mobile phase can quench the fluorescence of the molecule of interest. Most of the non-halogen-containing solvents used in HPLC can be used with fluorescence detection. However, dissolved oxygen or other impurities in the eluent can cause quenching. The solvent polarity and the pH of the mobile phase can also affect the fluorescent process if they influence the charge status of the chromophore. For example, aniline fluoresces at pH 7 and at pH 12, but at pH 2, where it is cationic, it does not fluoresce. Table 3.5 shows wavelength selections for some common LC-fluorescence applications.30... 

Figure 18 Effect of nonionic detergent (Triton X-100) on benzydamine N-oxygenation (FMO) and N-demethylation (CYP) by human liver microsomes. Benzydamine (500 pM) was incubated with pooled human liver microsomes (1.0 mg protein/mL) in tricine buffer (50 mM, pH 8.5 at 37°C) with or without Triton X-100 [1% (v/v)]. Reactions were initiated by the addition of an NADPH-generating system and stopped after 10 minute by the addition of an equal volume (500 pL) of methanol. Precipitated protein was removed by centrifugation, and an aliquot (25 pL) of the supernatant fraction was analyzed by HPLC with fluorescence detection. Abbreviations FMO, flavin monooxygenase CYP, cytochrome P450.

In case of SECM measurement of the cell activity, the reduction current of oxygen was detected. The tip was a platinum microdisc electrode with diameter of 5 pm. It was positioned 25-30 pm over the cells. The SECM measurements show immediately the action of respiration activity halting species while the fading of the fluorescence intensity change reflects the increase of membrane permeability resulted by exposure to toxic chemical. The less invasive nature of the SECM makes it a well applicable tool for testing the effect of different species on the cellular respiration. 

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

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