Application Of Structure-Matched Phthalocyanines Associate As A Red-Emitting Fluorescent Probe In The Determination Of Heparin At Nano-Gram Level

Heparin, a polyanionic bio-polysaccharide, has important clinical values. The development of simple, rapid and specific method for the detection of heparin has been being an attractive topic in the last decades. According to the principle of molecular association, a phthalocyanine-based ion-associate fluorescent probe was prepared, followed by the establishment of a novel fluorimetry for the determination of heparin with high specificity at nanogram level. Tetrasulfonated aluminum phthalocyanine (AlS4 Pc) , which emits strong red fluorescence is negatively charged, was found that its fluorescence could be dramatically quenched by a structure-matched cationic copper phthalocyanie (Alcian blue 8 GX) bearing a same parent structure and oppositely charged. Because of their highly matched molecular structures the two phthalynines, strong association is easy to occur through intermolecular force, leading to the formation of none-fluorescent associate. Based on this founding, a fluorescent probe of AlS4 Pc-Alcian blue 8 GX associate was developed in this study. Screening experiments on carbohydrates showed that the fluorescence of AlS4 Pc-Alcian blue 8 GX recovered when polyanionic bio-polysaccharide existed. The fluorescence recovery is particularly significant in the presence of heparin. We believe that this phenomenon could be attributed to the presence of a large number of sulfonate anion on the skeleton of heparin. The polyanionic structure leads to strong competitive binding to Alcian blue 8GX with AlS4 Pc, resulting in the dissociation of AlS4 Pc from the associate, and the fluorescence of the system restored. Based on the above findings, a highly sensitive and specific method for the determination of heparin by fluorescence enhancement was established. The molecular spectra ( fluorescence and absorption spectra) of the system were investigated to deduce the mechanism of the formation of associate and the fluorescence recovery. The reaction parameters (including pH, reaction temperature, reaction time, usage of AlS4 Pcand Alcian blue 8 GX) were optimized. Under the optimal conditions, the linear range of calibration curve is 6. 0 similar to 600. 0 ng.mL(-1), and the detection limit is 5. 7 ng.mL(-1). A facile pretreatment method employing polar organic solvent as precipitant was developed to avoid the deviation in the detection of practical samples. In addition, the interference behavior of foreign substances on the determination of heparin was investigated comprehensively, which has made up for the deficiency in literatures. The proposed method has been applied to the determination of practical sample (heparin sodium injection) with satisfactory results. This study expends the application of fluorescent phthalocyanines as molecular optical probes in analytical sciences.