Comparison of a series of hydrophilic squaraine and cyanine dyes for use as biological labels

Abstract

Unlike cyanine dyes, which are widely used as fluorescent probes and labels for biomedical applications squaraine dyes are less investigated. A series of monoreactive, water-soluble, squaraine dyes with two aromatic sulfo groups and up to 3 sulfobutyl groups was synthesized and the spectral properties of these dyes were compared to dicarbocyanines of identical structure. Compared to the cyanines in aqueous solutions the squaraine dyes absorb and emit at shorter wavelengths (630–636 nm/639–645 nm vs. 647–653 nm/665–672 nm), have higher molar absorptivities (284,000–333,000 M−1 cm−1vs. 242,000–260,000 M−1 cm−1), lower fluorescence quantum yields (4.3–9.4% vs. 27–32%) and lower fluorescence lifetimes (0.2–0.3 ns vs. 1.0–1.2 ns) but the quantum yields and lifetimes substantially increase when bound to proteins (Bovine Serum Albumin (BSA) or antibodies, immunoglobulin G (IgG)). Squaraines with two aromatic sulfo groups show no aggregation tendency up to concentrations of 2 × 10−4 M while the corresponding cyanine dye is free of aggregation up to 5 × 10−4 M. The increase in the number of sulfobutyl groups bears a strong influence on the aggregation tendency of both dye classes upon covalent labeling to BSA and IgG resulting in increased quantum yields and lifetimes of the protein conjugates. Compared to cyanines, squaraine dyes exhibit higher photostabilities and much higher sensitivity of the quantum yields and fluorescence lifetimes toward the microenvironment and are therefore better suited as fluorescence sensors.