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Cell analysis by optochemical sensing represents large and important niche in life and biomedical sciences. We present advanced multi-modal, multi-analyte sensing platform and dedicated materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of the different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of the ratiometric version of the pH sensor, assessment of photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration of sensor performance in Oxygen Consumption Rate (OCR) and Extracellular Acidification (ECA) measurements with relevant cell models. The main outcomes include the elaboration of the structure-function relationships for this biosensor system, development of the self-referenced tandem O2/pH and OCR/ECA sensing system which enables calibration-free operation, demonstration of the sensor operation on common plastic substrates in cell analysis, benchmarking against the existing platforms and real-life experiments with cells. This new sensing platform shows potential for wide practical use.

Conjugates of 17α-substituted testosterone (1 and 2) and 17β-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17β-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.

A new approach to functionalize porphyrinoids was developed based on the Vilsmeier formylation followed by the azine formation and a new class of the compounds namely azines of porphyrinoids was obtained. Variously substituted azine porphyrins, chlorins and their dyads were synthesized, and their structural and electronic parameters were studied with X-ray diffraction analysis, electronic absorption and emission spectra, electrochemistry and DFT calculations. Azine represents a very peculiar bridge with an unclear conjugation ability affecting optical properties of chromophores. This work was aimed to reveal azine properties and the influence of the azine bridge on the chromophores linked. The results of investigation allowed to conclude that azine bridge can partly be regarded as a conjugation switch with a high threshold level of switching, where limited conjugation in the ground state can be greatly increased at higher energy states of the azine molecule.

The previously unknown transformation of coproporphyrins to coprochlorins was achieved via reduction of coprohemins I and II with sodium in isoamyl alcohol to give the corresponding novel coprochlorins I and II. Preliminary mechanistic studies using DFT calculations suggested a reduction pathway leading first to the porphodimethene followed by rearrangement to the chlorin.

Synthesis of 13′[(cholest-5-en)-3β-yloxyethoxycarbamoyl]-chlorin e6 starting from methylpheophorbide and 3β(2-hydroxy)-ethoxycholest-5-ene is presented, as well as the preparation of related copper complex. Both conjugates obtained may be simply incorporated in phosphatidyl choline vesicles.

Several phosphorescent platinum(II) and palladium(II) complexes of tetrapyrrole dyes are prepared in an attempt to improve the compatibility with red laser diodes. The nature of meso-substituents in benzoporphyrin macrocycle is found to affect mainly the position of the Soret band and has little influence on the Q-bands. In rigid polymeric matrices these dyes possess more favourable photochemical properties than corresponding meso-tetraphenyltetrabenzoporphyrins. Platinum(II) chlorins show good compatibility with 635 nm laser diode and 632.8 line of He–Ne laser, and have moderate brightness with emission yields ∼2%. Photophysical properties and photostability of these dyes is assessed and compared with the known NIR-emitting oxygen indicators.

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.

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.

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.

New sensor technology and materials for luminescence-based oxygen sensors are described, which utilise microporous light-scattering support in sensor fabrication. Such materials display a number of new features and advantages over conventional oxygen sensor membranes, such as, significantly increased levels of luminescent signals obtainable from the sensor, better mechanical, physical and optical properties and a high capacity for loading biomaterial. The new oxygen sensor technology enables simple and robust procedures of sensor fabrication, as well as saving of the sensing materials, bringing down sensor photobleaching, and improvement in reliability of measurements and signal resolution. Biosensors developed on the basis of the above luminescent oxygen-sensitive materials used as chemical transducers, are described. Direct immobilisation of oxidase enzymes of the oxygen sensor membranes allows simple assays of metabolites. In particular, new batch-type enzyme biosensor for glucose has been investigated, using a new heterogeneous assay format. The latter allows simple, fast and highly reproducible analysis of important metabolites.

Protomeric forms of the porphyrin-ketones (or oxo-chlorins) are investigated by optical spectral methods. The main spectral forms of octaethylporphine-ketone and coproporphyrin-ketone tetramethyl ester appearing in organic solvents in the presence of trifluoroacetic acid, in SDS micellar solution and in sulfuric acid, namely, the free base, monocation, dication and trication, are identified and described in detail. Their mutual transformations are shown to proceed differently for the two dyes. The possibility of protonation of the macrocycle is also proven for the porphyrin-ketone complexes. Two new spectral forms of platinum(II) and palladium(II) complexes of the porphyrinketones appearing in organic solvents on addition of TFA are described. Possible mechanisms of protonation, including formation of oxonium ions and carbocations, are discussed for these dyes.

A set of luminescent dyes, namely the porphyrin-ketones and their zinc(II), platinum(II) and palladium(II) complexes, are described. The probes are derived from a partially oxidized porphyrin ring and have been studied by absorption and luminescence spectroscopy including time-resolved measurements. The metal-free porphyrin-ketones display strong and pH-dependent fluorescence due to a protolytic equilibrium that exists between the free base and the dication. The zinc(II) complexes also give strong fluorescence along with a weak long-decay emission at room temperature which are attributed to delayed fluorescence and phosphorescence, respectively. Platinum(II) and palladium(II) complexes exhibit room-temperature phosphorescence without any detectable fluorescence. Compared to existing porphyrins, the new dyes display a considerably longwave-shifted luminescence along with substantially improved (photo)chemical stability. They are considered to be promising probes for very near-infrared luminescence studies, and to be particularly useful for applications such as labelling of biomolecules, pH-sensing, and detection of quenching species such as molecular oxygen.

Reaction of various trimethyl(porphyrinylmethyl) ammonium iodides (generated in situ from the corresponding meso -dimethylaminomethylporphyrins and iodomethane) with triphenylphosphine afforded triphenylporphyrinylmethylphosphonium iodides which, when subjected to mild basic hydrolysis, gave meso -methylporphyrins in high yields.

Vilsmeier reaction of mono-Ni 1,2-ethanebis(octacthylporphyrin) favoured 10-substitution. Functional group intcrconvcrsions led to pure 10-dimethylaminomethyl, -azomethine, -methoxymethyl, and -formyl derivatives. Demetallation gave a range of labile 10-substiluted free base dinners. Oxidation of the mono-Ni compounds in AcOH gave the corresponding trans-ethylene mono-Ni derivatives, while the free bases were all converted to 10'-formyl-1,2-cthylenebis(octaethylporphyrin) under the same conditions.

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.

A new cis-form of ethylene-bridged octaethylporphyrin dimer 3a has been obtained by isomerization of the corresponding trans-isomer 2a . On the basis of spectral data the structure of 3a was assigned to be close to a “face-to-face” orientation. Reflecting the peculiar structure of 3a UV-vis and 1 H NMR spectra are remarkably distinguished from those of ordinary porphyrins.