Near-infrared probes: design and applications

Abstract

Near-Infrared (NIR) absorbing chromophores have been valuable in analytical and bioanalytical chemistry. NIR probes and labels have been used for several applications, including solvent polarity, hydrophobicity, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 nm) is more advantageous for the bioanalytical chemist due to the inherently lower background interference and the high molar absorptivities. NIR dyes can be used as simple probes to investigate biomolecule properties or just simply to detect the presence of biomolecules. Another typical application is the use of NIR fluorophores as labels. In these applications covalent labeling is the preferred method but it requires NIR dyes with appropriate reactive moieties. Due to the hydrophobic nature of NIR chromophores non-covalent labeling may be a viable alternative. For this purpose novel bis(carbocynines) have been developed in our laboratories. These dyes form intramolecular H-aggregates in polar solvents, even at very low concentrations. Spectral properties of this intramolecular dimer greatly depend on the properties of heterocyclic moieties and the length, the location and/or flexibility of the connecting chain. This form of the dye can be described as a clamshell complex with two interacting hydrophobic carbocyanine moieties. This intramolecular H-aggregate has a low extinction coefficient and fluorescence quantum yield. Upon opening the clamshell that can be facilitated by changing microhydrophobicity (i.e., binding to biomolecules) the H-and D- bands are decreased and the monomeric band is increased, with concomitant increase in fluorescence intensity. The main analytical utility of these bis(carbocyanines) is that the free dye (i.e., not complexed to an analyte) has negligible fluorescence in a typical aqueous buffer environment. Examples of different applications of these bis(carbocyanines) are given including forensic applications.