We describe the synthesis and spectral characterization of two reactive long-wavelength fluorescence labels (Sq635-m and Sq635-b), having either one or two N-hydroxysuccinimidyl esters. Both are squaraine derivatives and consist of a cyanine-type chromophore and a central squarate bridge. To improve water solubility, we introduced two sulfonic acid groups into the heterocyclic ring systems, and for covalent attachment to proteins, a reactive N-hydroxy-succinimide ester (NHS ester) was synthesized. The squaraine markers exhibit low quantum yields in water (phi = 0.15) and high quantum yields (phi = 0.6-0.7) when bound to proteins. The absorption maxima at 635 nm in water and at approximately 645 nm when bound to proteins allow excitation with commercially available diode lasers. The detection limit of a representative squaraine dye in blood was estimated to be half that of a commonly used fluorophore.

No abstract

We describe a series of new long-wave absorbing and fluorescing cyanine dyes and labels (based on a general logic for the design of such dyes), their spectra, covalent and noncovalent linkage to proteins, their use in single molecule detection (SMD) and as donors and acceptors, respectively, in fluorescence resonance energy transfer studies. The new labels represent water-soluble and reactive fluorophores whose quantum yields increase substantially if noncovalently or covalently bound to proteins. Due to their strong absorptions between 550 and 700 nm they are excitable by light-emitting diodes or diode lasers. Their high absorbances (epsilon around 100,000) and adequate fluorescence quantum yields (phi up to 0.68 if bound to proteins) along with their availability as reactive NHS esters make them viable labels for proteins and oligomers, e.g. in context with SMD or fluorescence energy transfer immunoassay which is demonstrated for the system HSA/anti-HSA.