The Application Of New Probes In Optical Sensors And Biological Analysis

Fluorescence measurement is one of the important detecting technology which is fast, highly accurate, sensitive. With the rapid development of the instruments and technologies including laser, microprocessor and electronics, the fluorescence measurement was greatly promoted in industry, agriculture, medicine, health, biology and other fields. The properties of the probes play an important role in fluorescence measurement. The probes can directly contact with the samples in homogenous phase. However, the tunable factors are limited in homogeneous phase so that the selectivity and detection limits are often difficult to optimize. If the probe was incorporated into the optical sensors, it will avoid contaminating the samples and the properties of sensors will be improved by optimize the sensing components. This thesis is divided into three parts to study the application of new fluorescent probes in optical sensor and biological analysis.(1) Highly fluorescent probes based on boron-dipyrromethene functionalized with a phenylboronic acid group (BODIPY-PBAs) were used to detect monosaccharides. The emission intensity of fluorophores increases when binding to the analytes with diol groups and form boronic esters at fixed pH. These compounds can detect monosaccharides in the concentration range of 0.1 to 100 mM. On the other hand, glycogen was found to quench the fluorescence of BODIPY-PBAs in aqueous solution due to the self-quenching of the fluorophores after attaching in the extensively branched and compact glucose polymer, further addition of D-fructose to the solution can release the fluorophores from the polymer and the fluorescence regains. The BODIPY-PBA fluorophore has been applied in polymeric optodes containing anion-exchangers to perform repetitive measurement. Such sensors respond to different monosaccharides in the range of 0.1 to 100 mM and demonstrate an improved selectivity toward D-fructose over other saccharides compared to the results obtained from homogeneous assay.(2) A novel hydrophobic probe based on BOD IP Y fluorophore 8-nitrophenyl-3,5-dipiperidin-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (NPBDP), to which two piperidine groups were attached by using facile synthetic method was used to detcte and image the intracellular lipid droplets by fluorescence microscopy. NPBDP has the absorbance maximum at 618 nm and the long wavelength emission peak at 677 nm in cyclohexane because the piperidine units linked to 3,5 positions of the BODIPY core and the conjugation of the electron pair on nitrogen atom with the bodipy core leads to nearly 160 nm red shift of the emission wavelength compared to the unsubstituted compound. Compared to the commercial lipid probe DiO in homogeneous phase, we found the fluorescence emission intensity of NPBDP in cyclohexane is 127 times of in ethanol under the same experimental conditions while the ratio of DiO in cyclohexane to ethanol is only 1.6. In lipophilic plasticized membranes with different polarity plasticizers bis (2-ethylhexyl) sebacate (DOS), dibutyl phthalate(DBP), dimethyl phthalate(DMP), tripropyl phosphate(TPP),2-nitrophenyl phthalate(DMP), tripropyl phosphate(TPP), the selectivity of NPBDP to less polarity of plasticizer is higher than DiO. Furthermore, the red emission can be observed in the lipid by confocal microscope, and the fluorescence became stronger for the thicker lipid layer that entraps more NPBDP. Finally, the fluorescence imaging and spectrum of NPBDP (10 uM) loaded-MCF7 cells show numerous red punctate spots indicating the intracellular lipid droplets bounded by cell membranes, NPBDP gave a much improved signal-to-noise ratio and no fluorescence in cytoplasmic matrix was observed. After incubation in the medium containing NPBDP for one week, MCF7 cell still displayed similar localization phenomenon and biological activity.(3) We rationally designed an aza-bodipy-BA as a new near infrared and ratiometric fluorescent probe for H2O2 by modification of the fluorophoric core at 3 and 5 position, so that the phenyl boronic acid group is conjugated to the core. The change on the boronic acid to phenol by H2O2 results in the different absorbance/excitation energy level, which gives ratiometric changes on the fluorescent spectrum. Aza-bodipy-BA exhibited high quantum yield and excellent photo stability. The absorbance at 655nm only changed 1% upon upon continuous irradiation of mercury lamp (135.9 mWcm-2) in Olympus BX53 microscope at room temperature while the fluorescence intensity changed 3.2% at 687nm. The boronic acid functionized aza-borondipyrromethene dye (aza-bodipy-BA) exhibits high selectivity toward hydrogen peroxide. The pH response of aza-bodipy-BA indicated that the dye is pH independent form in acidic sample till pH 7.5, which makes it quite useful for intracellular imaging and measurement. The dye emits at 687 nm upon the excitation of 652 nm. When adding different amount of H2O2, the emission peak was red-shifted gradually to 727 nm. Compared to the H2O2 response in homogenous, the response time and detection limit were decreased in membrane phase. The ratiometric fluorescence measurement in the near-infrared region allows the quantitative evaluation of the intracellular H2O2 level. The corresponding H2O2 concentration which is added into the cell is approximately 100μM when stimulating by 5μg/ml phorbol myristate acetate (PMA).