The enhancement effect of CaCl2 on solid substrate room temperature phosphorescence of polyhalogenated derivatives of fluorescein and application in immunoassay

It was found that the solid substrate room temperature phosphorescence (SS-RTP) signal of some polyhalogenated derivatives of fluorescein (including eosin5-isothiocyanate) on a polyamide membrane can be enhanced obviously when an external inorganic salt was introduced into the system. The enhancement effect (RTP intensity and the ratio of signal to noise) of CaCl2 was much more notable than those of typical heavy atom perturbers in phosphorimetry, such as Pb(AC)2 and Hg(NO3)2, etc. According to that, a solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) with eosin5-isothiocyanate as a marker and CaCl2 as an enhancer for the determination of human complement 3 was developed. On a polyamide membrane, SS-RTP signals (λex/λem = 532 nm/685 nm) of the immune complex are in linear correlation to the amount of human C3 in the range of 0.156-5.00 pg/spot (the sample volume was 0.4 μL/spot, corresponding concentration was 0.391-12.5 ng/mL). The regression equation of working curve can be expressed as ΔIp (ΔIp = Isample − Iblank) = 1.57 + 0.633 mC3 (pg/spot), correlation coefficient r = 0.998. The detection limit calculated as 3 Sb/k is 0.12 pg/spot. The method was directly applied to the determination of human complement 3 in human serums with satisfactory results. These results proved sufficiently that eosin-ITC is a good phosphorescent marker, while CaCl2 was used as the enhancer. Obviously, the results will expand the application area of these luminescent molecules, and using CaCl2 instead of Pb(AC)2 as a phosphorescence enhancer is more favorable for environment.