Ratiometric fluorescent detection of heparan sulfate in human plasma and serum using peptide-based fluorescent probes

The level of heparan sulfate (HS) in human blood acts as a biomarker for several diseases, making the development of sensitive and selective detection methods increasingly important. Until now, there has been no ratiometric fluorescent detection method for sensing HS in blood samples. In this study, we report a novel ratiometric fluorescent detection method for HS in blood samples using two peptide-based fluorescent probes: probe 1, containing six Arg residues, and probe 2, containing two Arg residues and four Lys residues. Both probes, excited with visible light at 430 nm, exhibited ratiometric fluorescence responses to HS in human plasma solutions. However, probe 1 exhibited a highly sensitive ratiometric response to HS at nanomolar concentrations (0-300 nM) in 50 % human plasma samples, whereas probe 2 exhibited ratiometric responses at nanomolar concentrations (0-800 nM) in plasma samples containing up to 30 % human plasma. Probe 1 also showed a highly selective ratiometric response to HS over heparin and other biological competitors in human plasma samples. The detection limit of probe 1 was determined to be 14.23 nM in 50 % human plasma samples. Structural analysis of the developed fluorescent probes revealed that the presence of six guanidine groups, rather than amino groups, significantly enhances HS detection in blood samples by minimizing non-specific binding to biomolecules. This work represents the first approach to quantifying HS levels in human serum and plasma samples using ratiometric fluorescence techniques, providing a promising tool for disease biomarker identification and clinical diagnostics.