Fluorescent probe of rhodamide-fluorescein type for pH determination
Fluorescent probes of fluorescein and rhodamine type are often used in the study of various biological processes which are accompanied by change of intracellular pH. These biological processes can include nucleic acids, specific proteins, etc. Fluorescein provides intensive green fluorescence (excitation/emission 495/512 nm) at alkaline and neutral pH. On the other hand, rhodamine then exhibits intensive red fluorescence (excitation/emission 563/572 nm) at acidic pH. Direct connection of both indicators then allowed determination of change in fluorescence of fluorescein and rhodamine at two excitation/emission wavelengths. The ratio of intensities of fluorescent maxima is then linear in certain area of pH.
The rhodamine and fluorescein type indicators are popular especially due to their good biocompatibility and high sensitivity at physiological conditions. The promising potential of probes included both fluorescein and rhodamine was predestined by described system in which fluorescein and rhodamine was linked through the diethylamine linker. This compound was successfully used for pH determination in mitochondria. We have developed indicator included both fluorescein and rhodamine having two excitation maxima at 494 nm and 566 nm which exhibit emission at 515nm and 585 nm. The ratio of emissions is dependent on change of pH and is linear in range of 6.6 – 7.8 pH.
The main advantages of our developed system are the possibility of pH determination with high accuracy reaching the values 0.1 pH, the independence of measured values on amount of indicator, good chemical stability and the ability to penetrate to living cells. The main disadvantage of already existing compounds is especially their complicated and time wasting synthesis which does not allowed the effective evaluation of the most suitable substrate. On the other hand, the mentioned disadvantage is eliminated in our system due to use of solid phase synthesis.
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc