Antonín Svoboda

Kinetics and efficiency of excitation energy transfer from chlorophylls, their heavy metal-substituted derivatives, and pheophytins to singlet oxygen

Time-resolved measurements of the singlet oxygen infrared (1269 nm) luminescence were used to follow the kinetics and efficiency of excitation energy transfer (EET) between chlorophyll (Chl) derivatives and oxygen in acetone. The studied pigments were Mg-Chl a and b and their heavy metal (Cu(2+) and Zn(2+))-substituted analogues, as well as pheophytin (Pheo) a and b. The efficiency of EET from chlorophyll to oxygen was highly dependent on the central ion in the pigment. Cu-Chl a and Cu-Chl b had the lowest efficiencies of singlet oxygen production, while Pheo a had a higher one, and Zn-Chl a had a similar one compared to Mg-Chl a. Also the side chain (position C-7, i.e. Chl a vs. Chl b) influenced the efficiency of singlet oxygen formation. In the case of square-planar complexes like Cu-Chl and Pheo, EET was more efficient in the Chl a derivatives than in those of Chl b; the opposite effect was observed in the case of the five- or six-coordinated Mg-Chl and Zn-Chl. As for the lifetime of the Chl triplet state, the most striking difference to Mg-Chl again was found in the case of Cu-Chls, which had much shorter lifetimes. Furthermore, the central ion in Chl affected the physical quenching of singlet oxygen: its efficiency was decreasing from Mg-Chl through Zn-Chl over Cu-Chl to Pheo. The results are discussed in the context of the oxidative stress accompanying heavy metal-induced stress in plants.

Phosphorescence of singlet oxygen and meso-tetra(4-sulfonatophenyl)porphin: time and spectral resolved study

Highly sensitive spectroscopic experimental setup was built to detect infrared luminescence with both time and spectral resolution. In this contribution, phosphorescence of meso-tetra(4-sulphonatophenyl)porphin, which is used as a photosensitizer for photodynamic therapy of cancer, and emission of singlet oxygen were studied. Two-dimensional matrices of data (counts as a function of time and wavelength) were obtained. From these matrices, 1.7 μs lifetime of the photosensitizer triplet state and the same rise-time of the singlet oxygen emission were resolved. Further, 3.7 μs lifetime of singlet oxygen phosphorescence was determined.

Luminescence Study of Singlet Oxygen Production by Meso-Tetraphenylporphine

The research in the field of the photodynamic therapy of cancer (PDT) is focused on a development of photosensitizers exhibiting high quantum yield of singlet oxygen production. Direct time-resolved spectroscopic observation of singlet oxygen phosphorescence can provide time constants of its population and depopulation as well as photosensitizer phosphorescence lifetime and relative quantum yields. In our contribution, a study of time and spectral resolved phosphorescence of singlet oxygen photosensitized by meso-tetraphenylporphine in acetone together with the photosensitizer phosphorescence is presented. Time constants of singlet oxygen population and depopulation were determined at wide range of photosensitizer concentrations. The time constant of singlet oxygen generation (0.28 ± 0.01) μs is slightly shorter then the lifetime of photosensitizers triplet state (0.32 ± 0.01) μs. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (≈50 μs) decreases with increasing photosensitizer concentration. Therefore, the photosensitizer acts also as a quencher of oxygen singlet state, similarly to the effects observed in [A. A. Krasnovsky, P. Cheng, R. E. Blankenship, T. A. Moore, and D. Gust (1993). Photochem. Photobiol.57, 324–330; H. Küpper, R. Dědic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj.1572, 107–113]. Moreover, the increasing concentraion of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.

Parallel fluorescence and phosphorescence monitoring of singlet oxygen photosensitization in rats

The time- and spectral-resolved set-up for measurements of weak infrared luminescence of photosensitizers (PSs) and singlet oxygen using optical lightguides was used on skin of laboratory animals in vivo. Wistar rats with abdominal incisions treated by methylis aminolevulinitis (MAL) were used as a model. A control group of animals with abdominal incisions was also tested. Spectrally resolved fluorescence of the PS was acquired during the treatment from the same spot. The intensity and spectral profile of the fluorescence signal from the skin can be used to guide the detection setup to the investigated spots in the lesion. The rate of bleaching of Protoporphyrin IX band and appearance of a band of its photoproducts during the treatment can be characterized by the exposition ED, under which the latter becomes dominant feature in fluorescence spectrum. ED value differs statistically significantly between the normal skin and the lesion treated by MAL. No direct proportionality was found between the fluorescence signal and singlet oxygen production. Nevertheless, the strong fluorescence signal is necessary but not a sufficient condition for higher singlet oxygen production in vivo. ED value correlates rather well with production of singlet oxygen, but differently in lesion and normal skin. Lifetimes of singlet oxygen differ between spots outside and in the lesion. PS triplet state lifetimes exhibit weak difference between spots treated and untreated by MAL.

Light-induced TPP photoproduct formation in chloroform and protective role of lipids

In this contribution, the influence of lipids on excitation energy transfer from lipophilic photosensitizer tetraphenylporphyrin to oxygen was investigated in chloroform solutions of phosphatidylcholine as well as in bulk lipid. The excited states kinetics were examined in a wide range of lipid concentrations (from zero to the saturated concentration) by direct time- and spectral-resolved detection of weak near infrared phosphorescence of the photosensitizer (around 840 nm) and singlet oxygen (about 1278 nm). While photosensitizer triplet kinetics follows single-exponential decay with lifetime of 0.52 μs in pure chloroform, two distinct components with lifetimes of approximately 0.4 and 1 μs appear after phosphatidylcholine addition. Both the lifetimes exhibit shortening tendency with increasing lipid concentration. Relative weights of the two components depend on the lipid concentration. Singlet oxygen kinetics exhibit single-exponential rise with lifetimes roughly corresponding to the shorter components of photosensitizer decays while their decays require two exponentials. The lifetime of the longer component decreases with increasing concentration of lipid from (77.6 ± 1.3) μs at pure chloroform to (14.3 ± 1.1) μs at the saturated lipid concentration. The time-constants obtained in bulk lipid sample follow the above-mentioned trends. Tetraphenylporphyrin photoproduct formation under pulsed excitation in chloroform solutions was demonstrated. The quantum yield of singlet oxygen production of the photoproduct is lower than that of the tetraphenylporphyrin. It was shown that lipids prevent the singlet-oxygen mediated formation of TPP photoproduct, probably by efficient quenching of singlet oxygen. This quenching is justified by shortening of the longer component of singlet oxygen luminescence decays with increasing concentration of the lipid. Moreover, the lipids also quench triplet states of the photosensitizer.

SIMPLE LABORATORY-MADE AUTOMATED SEQUENTIAL INJECTION ANALYSIS (SIA) DEVICE. I. DESIGN AND TESTING OF THE HARDWARE COMPONENT

The ever increasing demand for analyses in clinical, agricultural, pharmaceutical, industrial, and process analytical control has led to the development of Sequential Injection Analysis (SIA) (1). Use of a flow pattern, rather than constant monotonous flow, used in Flow Injection Analysis, requires synchronisation of sample zone injection with the start of each flow cycle. Therefore, a system configuration must be conceived which will allow sample zone injection, reagent addition, mixing, measurement, and ejection of reacted mixture by a combination of forward and reversed flow steps. These steps may have different lengths, durations, and speeds, provided that they always follow the same pattern. A typical SIA set-up (Fig. 1) utilises a multiport selection valve (MSV) coupled with a pump (PP) which generates the main pulsation-less flow with variable direction and speed. A bi-directional syringe pump complies well with INSTRUMENTATION SCIENCE & TECHNOLOGY, 30(1), 13–20 (2002)

SIMPLE LABORATORY-MADE AUTOMATED SEQUENTIAL INJECTION ANALYSIS (SIA) DEVICE. II. SIA OPERATIONAL SOFTWARE BASED ON LABVIEW® PROGRAMMING LANGUAGE

ABSTRACT The paper presented deals with the development of an automated computer-aided sequential injection analysis (SIA) system and its application to laboratory automation. In Part I, hardware and technical parameters of the SIA system were described. Part II introduces original lab-developed operational software (FaFSIA), enabling one to control the cited SIA system and to process experimental data, including their presentation. The software, based on graphic programming language LabVIEW, is described in detail.