Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy

Streptavidin and antibodies were labeled with phosphorescent platinum and p alladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lif etime) and using ELISA assays to determine the retention of biological acti vity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, p latinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyr in-labeled streptavidin conjugates were evaluated for DNA in situ hybridiza tion oh metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not o nly in mammals but also in plants. A modular-based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and D NA targets in an autofluorescent environment. Higher image contrasts were g enerally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).