Activatable
contrast agents are of ongoing research interest because they offer low background and high specificity to the imaging target. Engineered sensitivity to
protease activity is particularly desirable because
proteases are critical
biomarkers in
cancer,
infectious disease, inflammatory disorders, and so forth. Herein, we developed and characterized a set of
peptide-linked cyanine conjugates for dual-modal detection of
protease activity via photoacoustic (PA) and fluorescence imaging. The
peptide-
dye conjugates were designed to undergo contact quenching via intramolecular dimerization and contained n
dyes (n = 2, 3, or 4) with n - 1 cleavable
peptide substrates. The absorption peaks of the conjugates were blue-shifted 50 nm relative to the free
dye and had quenched fluorescence. This effect was sensitive to
solvent polarity and could be reversed by
solvent switching from water to
dimethyl sulfoxide. Employing
trypsin as a model
protease, we observed a 2.5-fold recovery of the peak absorbance, 330-4600-fold fluorescent enhancement, and picomolar detection limits following proteolysis. The dimer probe was further characterized for PA activation. Proteolysis released single
dye-
peptide fragments that produced a 5-fold PA enhancement through the increased absorption at 680 nm with nanomolar sensitivity to
trypsin. The
peptide substrate could also be tuned for
protease selectivity; as a proof-of-concept, we detected the main
protease (Mpro) associated with the viral replication in
SARS-CoV-2 infection. Last, the activated probe was imaged subcutaneously in mice and signal was linearly correlated to the cleaved probe. Overall, these results demonstrate a tunable scaffold for the PA molecular imaging of
protease activity with potential value in areas such as disease monitoring,
tumor imaging, intraoperative imaging, in vitro diagnostics, and point-of-care sensing.