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Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.

Phosphorescent platinum(II)-coproporphyrin label (PtCP) was evaluated in post-PCR detection of nucleic acids by time-resolved fluorescence (TR-F) using three common formats. PtCP-labelled oligonucleotide primers and PtCP-dUTP were incorporated in a PCR to produce labelled amplified target −173 or 305 bp DNA. Alternatively, aminoallyl-dUTP was incorporated in a PCR and the product was subsequently labelled with PtCP. The resulting PCR mixtures containing labelled dsDNA were separated on 1.5% agarose gels and then analysed by ethidium bromide staining and by direct detection of PtCP label on a commercial TR-F plate reader Victor 2 (Perkin Elmer Life Sciences) used in scanning mode. In all cases label incorporation and high yields of amplified DNA were observed. Direct TR-F detection of PtCP-labelled DNA from a gel provided high sensitivity and signal to noise ratio, with limits of detection in the range of 9–22 pg for all three formats. The sensitivity achieved with PtCP label was considerably better than that achieved with ethidium bromide staining (∼1 ng of dsDNA) or with conventional fluorescent label FITC. Neither the FITC label nor ethidium bromide staining interfered with PtCP detection, thus allowing multiplexed detection.

Spectral-luminescent properties and quenching behavior of the covalent conjugate of the platinum(II) complex of coproporphyrin-I and bovine serum albumin (PtCP–BSA) was investigated. Quenching of phosphorescence by sulfite was studied in detail in an acidic range as a function of pH. Strong dynamic quenching of the phosphorescence by sulfite occurred at pH below 4.0 which was attributed to the formation of sulfur dioxide. The quenching reached its maximal level at pH below 1.5, Stern–Volmer quenching constants being as high as 2000 M−1. A new flow-injection system for the determination of sulfite was developed based on the PtCP–BSA conjugate immobilized on a preactivated Biodyne ABC membrane and placed in a flow-cell, with an acidic carrier buffer and fiber-optic phosphorescent detector. Such a solid-state phosphorescent probe is quenchable by sulfite and allowed rapid on-line detection of sulfite with a detection limit of about 10 μM. Performance of the new system and possible interferences were investigated.

Phosphorescent polymer film compositions and phosphorescent Langmuir-Blodgett coatings based on platinum and palladium porphyrins were tested in the presence of nitrogen oxides (NOx). Dynamic quenching of phosphorescence by NOx was observed. It was accompanied by chemical interaction of NOx with the dye molecules which caused persistent decrease of the phosphorescence signal and irreversible degradation of the sensor. Two modes of NOx quantitation based on measurement of either phosphorescence lifetime or time drift of intensity were examined. For the phosphorescent polymer film compositions the analytical range of NOx was found to be 0-3.0% ( m m ) with a detection limit of 0.1%. For the LB-film coatings the sensitivity for NOx was about five times better: the range was 0-1.0% ( m m ) with a detection limit of 0.02%. Although for both systems the analytical range of NOx covered was poor for practical purposes, the data obtained are useful for evaluation of possible interferences by NOx in other applications, such as in oxygen and SO2 sensors. The possibilities of the realization of a dual-sensing concept with a single phosphorescent probe is discussed with respect to simultaneous measurement of NOx oxygen using polymer film compositions and NOx SO 2 using Langmuir-Blodgett coatings.