Lifetime-based photoacoustic probe activation modeled by a dual methylene blue-lysine conjugate

Activatable photoacoustic probes have a promising future due to their ability to provide high-resolution, high-penetration depth information on enzyme activity in vivo. Spectral identification methods, however, suffer from heterogeneous optical properties and wavelength-dependent light attenuation in tissue, thereby limiting the effective suppression of background noise signal. Our approach is predicated on probing the excited-state lifetime of a dual-labeled methylene blue (MB) probe that changes its lifetime from short to long upon cleavage. Recently, we have reported on the ability of our system to probe the long triplet lifetime of free MB monomers in solution and to differentiate between monomers and dimers based on their lifetime contrast. Here we introduce an improvement to our system that significantly increases the system sensitivity to fast changes, and reduces the minimum resolvable lifetime down to a few nanoseconds. We applied this method to probe the excited-state lifetime of a covalently coupled dual methylene blue-lysine conjugate (MB2K) in a mixed MB/MB2K solution. Preliminary results show that a stable dimeric bond is formed between the chromophores within the conjugate, and that this conjugate is statically quenched. Examination of the transient absorption of MB2K reveals it does not exhibit a triplet excited-state lifetime, suggesting that it undergoes a fast deexcitation process directly from the singlet state. Finally, we demonstrate how the transient photoacoustic lifetime signal can be used to selectively detect the presence of MB monomers while improving background noise suppression by differentiating the lifetime of free MB dye with other absorbing structures.