Determination of Bioactive Matter by Affinity Adsorption Solid Substrate–Room Temperature Phosphorimetry Based on Lectin Labeled with Self-ordered Ring of Eosin Y

Abstract

A new phosphorescent labeling reagent named self-ordered ring (ESOR) of eosin Y (E) was developed. And the application of the determination of bioactive matter by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) based on ESOR labeling lectin was studied. Results showed that pink and homogeneous ESOR could be formed by E on polyamide membrane (PAM) in the presence of cetyltrimethylammonium bromide (CTAB) and ammonia water. ESOR could emit strong and stable room temperature phosphorescence (RTP) signal of E in the presence of heavy atom perturber. Specific affinity adsorption (AA) reactions could be carried out between the products of concanavalin agglutinin (Con A), triticum vulgaris lectin (WGA) labeled with ESOR and alpha-fetoprotein variant (AFP-V), alkaline phosphatase (ALP), glucose (G), respectively. Not only did the products of the affinity adsorption reactions preserve good RTP characteristic of E, but also the DeltaIp (DeltaIp=Ip2-Ip1, Ip1 is the RTP intensity of blank reagent, Ip2 is the RTP intensity of sample) of these products was proportional to the content of AFP-V, ALP and G, respectively. According to the facts above, a new method of AA-SS-RTP for the determination of AFP-V, ALP and G was established, based on ESOR labeling lectin. Detection limits (LD) of this method were 0.040 fg spot(-1) for AFP-V, 0.045 fg spot(-1) for ALP and 0.090 fg spot(-1) for G. And it has been successfully applied to the determination of AFP-V in human serum as well as the survey and forecast of human diseases. This method had high sensitivity, good repeatability, long RTP lifetime and little background interference with lamdamaxem at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V by AA-SS-RTP based on Con A labeled with ESOR was also discussed.