Subjects of Study: processes of interaction of squaraines and polymethines with low-molecular-weight and high-molecular-weight bioanalytes and related processes of dye fluorescence characteristics changes. Objective of Study: to increase the sensitivity and reliability of bioanalytical methods using new fluoresccent reagents based on longwavelength squaraine dyes. Research Methods: absorption and fluorescence spectroscopy, fluorescence lifetime phase-modulation spectroscopy. Theoretical and Practical Results and Novelty: potential use of squaraines as bioanalytical reagents for fluorescent determination of low-molecular-weight and high-molecular-weight bioanalytes, especially using fluorescence lifetime as read-out parameter was demonstrated based on systematic analysis of spectral and fluorescent, photophysical and photochemical properties of new longwavelength squaraine dyes, their non-covalent complexes and covalent conjugates with biotin, oligonucleotides and proteins; based on analysis of the relation between spectral, photophysical and photochemical properties with molecular structure of squaraine dyes criteria of dye selection for development of methods for fluorescent determination of low-molecular-weight and high-molecular-weight bioanalytes were formulated; впервые were proposed methodological approaches for determination of oligonucleotides and low-molecular-weight antigens using only one squaraine dye, which fluorescence lifetime sensitive to microenvironment. Degree of implementation: Squaraine dyes K8-1340, K8-1351 and K8-1661 were proposed as the most bright, sensitive and stable bioanalytical reagents. Their availability was proved at the Institute for molecular biology and genetics NAS of Ukraine (Kyiv), Institute of problems of cryobiology and cryomedicine NAS of Ukraine (Kharkiv), and also at foreing companies Agilent (USA), Tecan (Austria), ReaMetrix, Inc. (USA), Dako (Denmark), Luminex Corp. (USA), Molecular Devices (USA), ISS (USA), Edinburgh Instruments (UK), Becton Dickinson (USA), Pierce Biotechnology (USA), Avanti Polar Lipids, Inc. (USA), Glen Research Corp. (USA) and others. The obtained fluorescent conjugates and developed methods of bioanalyt determination are utilized for the analysis and quantitive determination of proteins; for the studies of antigen-antibody interactions in immunology; for hybridization analysis, based on the interaction of the oligonucleotides complementory pairs; for fluorescent microscopy biological imaging and during other bioanalytical and medical tasks. Peptide conjugates of dyes K8-1610 and K8-1660 (ULight series) are used by the PerkinElmer company (USA, Canada) for commercial test-systems LANCE® Ultra for the determination of activity of the kinase enzymes. Scope of Application: Analytical Chemistry.
