Seta-633 - A NIR fluorescence lifetime label for low-molecular-weight analytes

We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR) dye, and its use as a fluorescent label to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter. In a model assay, we demonstrate that a biotinylated Seta-633 tracer binds to antibiotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 1.8-fold upon binding to a specific antibody (antibiotin, MW = 160 kDa), while the titration with bovine serum albumin (BSA) or nonspecific antibody does not result in a noticeable change in lifetime. This behavior is contrary to that of fluorescent tracers like Cy5 or Alexa 647, which typically exhibit much smaller lifetime changes upon binding to antibodies.