Synthesis of a new long-wavelength latent fluorimetric indicator for analytes determination in the DT-Diaphorase coupling dehydrogenase assay system

We synthesized a new long-wavelength latent fluorogenic probe BQC (1) to monitor DTD activity. The fluorogenic chemical transformation of BQC triggered by DTD in the presence of NADH is through a series of tandem reactions, DTD-catalyzed benzoquinone reduction, trimethyl-locks cyclization and intramolecular urea formation, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by menadione. The fluorescent response of BQC is insensitive to various biological thiol reductants. Furthermore, pro-fluorophore BQC is a sensitive fluorimetric indicator for analytes determination in the oxygen-insensitive DTD-coupled dehydrogenases assay by including NAD+ which will convert to NADH by reaction in the presence of analytes. This novel oxygen-insensitive assay demonstrates a good relationship in detecting 3-hydroxybutyrate and glucose-1-phosphate in 1–10 μM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.