Date of Award
Doctor of Philosophy in Chemistry - (Ph.D.)
Chemistry and Environmental Science
Lev N. Krasnoperov
Edgardo Tabion Farinas
New luminescent probes with enhanced brightness convenient for bioconjugation were synthesized and tested in major biological applications including the detection of nucleic acids and living cells. The first project used luminescent lanthanide ion probes that take advantage of hyper-sensitive acquisition of their long-lived emission signal in time- resolved mode, which avoids short-lived background fluorescence of the medium. Two model carbostyril fluorophores (cs 124-CH3 and cs 124-CF3) were modified by inclusion of two spacers (diamino and bi-phenyl) for the attachment of amine and thiol reactive cross-linking groups. In this project, time-resolved measurements are presented for spectroscopy and microscopy. The probes were synthesized, then characterized by UV/VIS, NMR, mass spectrometry and then validated in various biological applications. DNA detection using luminescently labeled molecular beacons hybridization probes was performed with sensitivity less then 1 pM, which is about 100 fold higher then that for conventional fluorescent probes. Further 30-fold increase in detection sensitivity was achieved by tethering multiple lanthanide probes to a carrier molecule, avidin. The resulting avidin conjugates were used for imaging of living cells employing time-gated fluorescent microscopy. Six thiol-reactive derivatives of the lanthanide ion probes with bromoacetamido- and maleimido- cross-linking groups were synthesized. It was found that maleimido-compounds displayed exceptional reactivity instantaneously coupling to thiols at physiological conditions at micromolar thiol concentrations. The second part of the project included the development of light-emitting diagnostic molecules for the detection of human microbial pathogens. The approach employs fluorescently labeled antibiotics, which retain high specificity towards pathogens and render them fluorescent after binding to their cellular targets. These studies present that the obtained derivative 7- aminoDDAO is ~2.8 fold brighter than its predecessor DDAO, with excitation and emission maxima located in the spectral area where body tissues are the most transparent (λem= 680 nm). Using near-infrared emitting dyes, this approach presents non-invasive tomographic detection of the pathogens in the living body, thereby enabling swift and efficient therapies. The desired NIR probes were synthesized and validated in a collaboration study by detection of fungal infections in mice.
Wirpsza, Laura A., "Development of luminescent probes for ultrasensitive detection of biopolymers, their complexes, and living cells" (2012). Dissertations. 353.