Seta-633 - A NIR Fluorescence Lifetime Label for Low-Molecular-Weight Analytes

Abstract

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.

Squaraine Compounds: Tailored Design and Synthesis towards a Variety of Material Science Applications

“…[58] and with biotin to monitor the biotin/antibiotin interaction by fluorescence lifetime analysis. [59] Welder et al [60] described the interaction of 3-oxosubstituted squaraines in the presence of various proteins. They concluded that both the symmetrical and unsymmetrical squarylium dyes show enhancement of fluorescence intensity upon noncovalent interactions with proteins such as HSA, β-lactoglobulin A and trypsinogen.…”

Long-wavelength fluorescence lifetime labels

“…7), 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 are described in [108]. In a model assay, it was demonstrated that a biotinylated Seta-633 tracer binds to antibiotin with high specificity.…”

Long-Wavelength Probes and Labels Based on Cyanines and Squaraines

“…However, our own investigations show that the increase in photostability for Alexa Fluor 647 and its protein conjugates compared to Cy5 is not very pronounced ( Fig. 2) [17][18][19]. Both Cy and Alexa Fluor dyes have small Stokes shifts (20-30 nm).…”

“…Recently, SETA BioMedicals has developed a new near-infrared squarainebased label Seta-633, which can be used to study the interaction between lowmolecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647.…”

“…13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its [19] conjugates are more photostable as compared to Cy5 and Alexa 647. Other squaraine-type labels that show large, distinct lifetime changes after binding to proteins are listed in the Table 3 [115].…”