Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Unexpected photoluminescence of fluorinated naphthalene diimides.

Two new amino core-substituted naphthalene diimides (cNDIs) bearing fluorinated side chains have been synthesised. Steady-state and time-resolved fluorescence spectroscopy reveals unprecedented optical properties for the cNDIs with high quantum yields of ∼0.8 and fluorescence lifetimes of ∼13 ns in a range of solvents. These properties are apparent at the level of single molecules, where the compounds also show exceptional photostability under pulsed-laser excitation. Photon emission is remarkably consistent with very few long timescale (millisecond or longer) interruptions with molecules regularly undergoing >10(7) cycles of excitation and emission. Intermittencies owing to triplet-state formation occur on a sub-millisecond timescale with a low yield of 1-2%, indicating that the presence of the fluorine atoms does not lead to a significant triplet yield through the heavy-atom effect. These properties make the compounds excellent candidates for single-molecule labelling applications.

Mixed alkoxy/hydroxy 1,8-naphthalimides: expanded fluorescence colour palette and in vitro bioactivity.

An efficient and functional group tolerant route to access hydroxy 1,8-naphthalimides has been used to synthesise a range of mono- and disubstituted hydroxy-1,8-naphthalimides with fluorescence emissions covering the visible spectrum. The dialkoxy substituted compounds prepared possess high quantum yields (up to 0.95) and long fluorescent lifetimes (up to 14 ns). The method has been used to generate scriptaid analogues that successfully inhibit HDAC6 in vitro with tubulin acetylation assays confirming that these compounds are more effective than tubastatin.

Elucidation of naphthalene diimide metallomacrocycles and catenanes by solvent dependent excimer and exciplex emission.

Metallomacrocycles and [2]-catenanes based on a leucine substituted naphthalene diimide ligand have been confirmed in solution through detection of excimer and exciplex emission. Comparison with the behaviour of the free ligand provides insight into the solution speciation of the metallosupramolecular complexes and their solvent dependent nature.

A fluorescent "2 in 1" proton sensor and polarity probe based on core substituted naphthalene diimide.

A new core-substituted naphthalene diimide (cNDI) compound that is sensitive to both H(+) and solvent polarity has been synthesised and characterised. Optical absorption and emission change upon protonation while emission quantum yield and decay kinetics of the unprotonated form vary significantly with solvent polarity due to delayed fluorescence.