Phthalocyanine dye as an extremely photostable and highly fluorescent near-infrared labeling reagent

Abstract

Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations in photostability. This compromises their performance in applications that require a photostable fluorescent reporting group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important for observing and tracking individual molecular events over extended period of time. We report in this paper an extremely photostable and highly fluorescent phthalocyanine dye, IRDye^TM 700DX, as a near-infrared fluorescence labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g. Alexa Fluor"R"680, Cy^TM5.5, Cy^TM7 and IRDye^TM 800CW dyes; and about 27 times more photostable than tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with IRDye^TM700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The IRDye^TM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in Western blot and cytoblot assays.