Quantitative Measurement Techniques For Singlet Oxygen From Photosensitization Based On The Detection Of Near-Infrared Luminescence And Probe Fluorescence

Singlet oxygen (’02) is believed to be the major cytotoxic species generated during Type-II photochemical reaction of photodynamic therapy (PDT). As PDT continues to develop and find more potential clinical indications, quantitative detection of1O2amount from photosensitization has become a critical challenge for PDT dosimetry study. In this dissertation, quantitative measurement techniques for1O2from photosensitization based on the detection of1O2near-infrared luminescence and fluorescence of1O2-specific probe were developed, respectively. The main research contents of this dissertation are as follows:Firstly, a new sensitive1O2near-infrared luminescence detection system combined with oxygen partial pressure and photosensitizer fluorescence monitor function was developed to directly measure the time-resolved1O2luminescence signal. The influence of pulse-height discrimination threshold on the1O2luminescence measurement was investigated by the developed1O2near-infrared luminescence detection system and the optimal threshold for our detection system was found for signal discrimination.Secondly, with the above developed1O2near-infrared luminescence detection system, the time-resolved1O2luminescence of photosensitizers in organic solvents, aqueous solution containing1O2quencher sodium azide (NaN3), bio-tissue phantom and aqueous solution with different oxygen partial pressure were measured, respectively. This allows the extraction of important information about the photosensitized generation of1O2and its decay at different environments. In particular, depending on the quencher or oxygen concentration, the rise or the decay time of the luminescence signal exhibit different meanings regarding the lifetime of1O2or triplet state of photosensitizers, which can be further confirmed by using laser flash photolysis or1O2quenching method. As a results, the triplet state lifetime of Rose Bengal (RB),1O2lifetime and rate constant of1O2quenching by NaN3were determined, respectively.Thirdly, the feasibility for quantitative measurement of1O2generation by using a newly developed1O2-specific fluorescence probe Singlet Oxygen Sensor Green reagent (SOSG) was firstly demonstrated. The kinetic analysis of the RB-sensitized photooxidation of SOSG was performed, and the quantitative measurement of1O2generation can be the determined from the initial reaction rate.Finally, the obtained1O2quantum yields of porphyrin-based photosensitizer HiPorfin, HMME and PsD-007in PBS solution by using SOSG were in good agreement with the values, which wereindependently determined by using direct measurement of1O2luminescence. These results firstly demonstrate that the quantitative measurement of1O2generation using SOSG can be achieved by determining the initial reaction rates with an appropriate measurement protocol.In conclusion, the sensitive1O2near-infrared luminescence detection system can be used to directly measure the1O2luminescence intensity and lifetime in different microenvironments. The1O2quantitative analysis technique with the fluorescence probe SOSG provide an alternatively effective method for determing the’02quantum yields of the newly developed photosensitizers.