The use of the NIR spectral region (650-900 nm) is advantageous due to the inherently lower background interference and the high molar absorptivities of NIR chromophores. Near-Infrared (NIR) dyes are increasingly used in the biological and medical field. The binding characteristics of NIR dyes to biomolecules are possibly controlled by several factors, including hydrophobicity, size and charge just to mention a few parameters. Binding characteristics of symmetric carbocyanines and found that the hydrophobic nature of the NIR dye is only partially responsible for the binding strength. Upon binding to biomolecules significant fluorescence enhancement can be observed for symmetrical carbocyanines. This fluorescence amplification facilitates the detection of the NIR dye and enhances its utility as NIR reporter. This manuscript discusses some probe and marker applications of such NIR fluorescent dyes. One application discussed here is the use of NIR dyes as markers. For labeling applications the fluorescence intensity of the NIR fluorescent label can significantly be increased by enclosing several dye molecules in nanoparticles. To decrease self quenching dyes that have relatively large Stokes’ shift needs to be used. This is achieved by substituting meso position halogens with amino moiety. This substitution can also serve as a linker to covalently attach the dye molecule to the nanoparticle backbone. We report here on the preparation of NIR fluorescent silica nanoparticles. Silica nanoparticles that are modified with aminoreactive moieties can be used as bright fluorescent labels in bioanalytical applications. A new bioanalytical technique to detect and monitor the catalytic activity of the sulfur assimilating enzyme using NIR dyes is reported as well. In this spectroscopic bioanalytical assay a family of Fischer based n-butyl sulfonate substituted dyes that exhibit distinct variation in absorbance and fluorescence properties and strong binding to serum albumin as its sulfonic acid moiety is modified to less water soluble moiety was identified. In polar solvents, these water soluble compounds are strongly fluorescent, however form the less soluble aggregated species with virtual loss of fluorescence when the sulfonate groups are cleaved by enzymatic activity to form the corresponding straight chain alkyl aldehyde derivatives. To achieve this conversion in vitro photo-reduced riboflavin mononucleotide (FMN) with a glucose/ glucose-oxygenase oxygen scavenging system was utilized. The reduced FMN serves as a key substrate in the enzymatic desulfonation. Once the FMNH2 was produced the desulfonation reaction was characterized by using Laser Induced Fluorescence Capillary Zone Electropheresis (LIF-CZE). This method can be utilized as an assay to detect the enzyme activity in vitro with the possibilities of in vivo applications.

Date Published: 21 February 2013
PDF: 12 pages
Proc. SPIE 8596, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V, 859604 (21 February 2013); doi: 10.1117/12.2011595

Published in SPIE Proceedings Vol. 8596:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V
Samuel Achilefu; Ramesh Raghavachari, Editor(s)