We describe two new fluorescence resonance energy transfer (FRET) compatible labels, their covalent linkage to oligonucleotides, and their use as donor and acceptor, respectively, in FRET hybridization studies. The dyes belong to the cyanine dyes, and water solubility is imparted by a phosphonate which represents a new solubilizing group in DNA labels. They were linked to amino-modified synthetic oligonucleotides via oxysuccinimide (OSI) esters. The studies performed include binding assays, determinations of molecular distances, homogeneous competitive assays, and limits of detection, which are in the order of 5 pmol/L for a 15-mer.

We describe the synthesis, purification, and spectral properties of new dyes and reactive labels. They absorb in the visible range between 450 and 700 nm and display analytically useful fluorescence. They were made amino-reactive by esterification with N-hydroxysuccinimide (NHS). The resulting oxysuccinimide (OSI) esters were covalently linked to the amino groups of human serum albumin (HSA) or certain DNA oligomers. Except for dyes 9 and 13, they contain one reactive group only in order to avoid cross linking of biomolecules. Labeling of amino-modified biomolecules was performed by standard protocols, and the labeled proteins and oligonucleotides were separated from the unreacted dye by gel chromatography using Sephadex G25 as the stationary phase in the case of proteins, and reversed-phase HPLC in the case of DNA oligomers. The dyes also have been used as donor–acceptor pairs in fluorescence energy transfer systems and in energy transfer cascades.

A novel immunosystem is described that exploits the effect of luminescence energy transfer from a luminescently labeled antigen to a fluorescent antibody. A luminescent ruthenium–ligand complex (D-455) with absorption/emission maxima at 456/639 nm, respectively, was employed as the donor label, and a squaraine-type cyanine label (636/655 nm), as the fluorescent acceptor label. Specifically, the system human serum albumin (HSA)/anti-HSA was studied. HSA was labeled with the donor dye D-455, and anti-HSA was labeled with the acceptor dye A-631. On formation of the antigen–antibody complex, energy transfer occurs. The radiationless energy transfer affects both the decay time of D-455 and the intensities of the emissions of both D-455 and A-631. The decay time of around 500 ns of D-455 allows frequency–domain measurements in the low kilohertz range and therefore can be based on the use of conventional optoelectronics. This also suggests gated measurements to be performed. The major difference from existing HSA immunosystems is the use of a slow decaying ruthenium–ligand complex as the donor and of a long-wave emitting cyanine acceptor dye having a high quantum yield and a decay kinetics that is governed by the rate of energy transfer from the slow decaying donor.

The design of fluorescent probes (and labels) is as challenging as it ever was. Such probes enable studies on the molecular dimensions and dynamics of even complex (bio)matter, but also bioanalytical and screening assays whose sensitivity can reach the single molecule level. The design of advanced labels for bioassays is paralleled by developments in (laser) fluorescence spectroscopy, opto-electronics and data processing. Light-emitting diodes (LEDs) and diode lasers (DLs) are particularly attractive light sources and we therefore have focused our research (a) on labels that are LED- or DL-compatible, and (b) on applications of such labels to various analytical formats.In this article, we give an overview of our recent activities in the following areas: (1) a general logic for designing fluorescent probes and labels; (2) new diode laser-excitable probes for non-cocalent protein detection; (3) diode laser-compatible amino-reactive covalent labels; (4) diode laser-assisted fluorescent single molecule detection of dyes and labeled proteins; (5) new labels for flow cytometric determination of HSA; (6) new DNA labels; (7) fluorescence resonance energy transfer gene assays; (8) reactive ruthenium ligand complexes as markers for bioassays; (9) diode laser-excitable fluorescent polymer beads; (10) polyaniline-coated nanobeads as fluorescent pH probes; (11) phosphorescent poly(acrylonitrile) nanospheres as markers for optical assays; (12) competititve binding of streptavidin to biotinylated nanobeads as studied by resonance energy transfer; (13) nanobeads as reference dyes in luminescent lifetime imaging using DLR; (14) phosphorescent nanospheres for use in advanced time-resolved multiplexed bioassays; (15) beads dyed with a europium-based label and excitable with the 405-nm diode laser; and (16) a europium(III)-based probe for use in oxidase-associated reactions.

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 100000) 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.

We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.

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.