Yin Nah Teo

We report a fluorescence-based method for the detection of acrylic acid, a high value chemical precursor of acrylate esters and polymers. Our method generates a fluorescent pyrazoline product in the presence of acrylic acid using photoactivated 1,3-dipolar cycloaddition with a diaryltetrazole-based probe, and is capable of detecting, as little as 36 ppb of acrylic acid in vitro and in vivo. We anticipate that this probe will be useful in high throughput screens for biosynthetic production of acrylic acid from biomass and can replace existing chromatographic methods of detection such as GCMS and LCMS, which are low throughput, time-consuming and require sample preparation.

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide–protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53–Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53–Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410–670 nm. We developed a new approach to antibody conjugation, using Huisgen–Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340–380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.

To explore a new modular metal ion sensor design strategy, fluorophores and ligands were incorporated into short DNA-like oligomers. Compound 1 was found to function as a selective sensor for Ag⁺ in aqueous buffer, where low micromolar concentrations of Ag⁺ induce a red-shifted, turn-on fluorescence signal. Experiments with HeLa cells show that 1 can penetrate cells and yield a signal for intracellular Ag⁺. This suggests a broadly applicable approach to developing sensors for a wide variety of cations.

DNA-scaffolded oligodeoxyriboside fluorophores (ODFs) were used as the reporters in turn-on sensing of enzymatic bond-cleaving activity. A tetramer ODF of pyrene deoxynucleosides displayed high quenching efficiency when conjugated via ester linkages with a dabcyl quencher, and yielded large signal increases with several enzymes in vitro and in intact human cells.