Syntheses of novel dipyrrylmethene-BF2 dyes and their performance as labels in two-photon excited fluoroimmunoassay

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia(TM) TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia(TM) TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia(TM) TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia(TM) TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.