Yin Nah Teo

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

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.

Polyfluorophores on a DNA Backbone: Sensors of Small Molecules in the Vapor Phase

Vapors of small molecules are under widespread study in natural signalling, environmental monitoring, industrial quality control, and in medicine.[1–3] In the last few decades, advances in chemistry and material science have enabled the development of chemical sensors of such volatile organics,[4–7] in which conjugated polymers and cross-reactive chemical sensor arrays are the most widespread sensor types. Among them, optical vapor sensing arrays based on luminescence (including fluorescence) and absorbance have become essential.[8–13] In the recent past, colorimetric detection of volatile organics using crossreactive chemical sensors have been demonstrated in multiple applications.[14–16] Although absorbance–based methods for vapor detection have shown good success, fluorescence-based methods may offer some advantages, such as high sensitivity and low background. For example, the detection of vapors of nitroaromatics with conjugated polymers and bio-polymers has showed very high sensitivity.[17–23] Notably, much of the

Selective Sensor for Silver Ions Built From Polyfluorophores on a DNA Backbone

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–polyfluorophore excimers as sensitive reporters for esterases and lipases

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.

Protease Probes Built from DNA: Multispectral Fluorescent DNA–Peptide Conjugates as Caspase Chemosensors†

The direct monitoring of enzyme activities is broadly useful in many fields, ranging from biochemistry to medicinal chemistry and biology.[1] A biological process often involves multiple enzymes, which can work independently or cooperatively to control a specific biological event. Monitoring their activities can provide markers of the progress of such a process (such as the cascade of caspase activity that occurs in apoptosis), and yield information about the mechanism and timing of the interacting species. The ability to track multiple targets in a single event or different processes simultaneously could greatly improve our basic understanding, and facilitate biological and clinical studies as well.

Studies of oligodeoxyfluorosides (ODFs) as FRET probes for DNA hybridization

Oligodeoxyfluorosides (ODFs) are a novel system of stacked, electronically interacting fluorophores built on the DNA scaffold. Here we describe early studies of these ODFs as potential universal FRET donors and as reporters of DNA hybridization.