Highly sensitive and selective biosensor for heparin detection with rhodamine B-labelled peptides as fluorescent bioreceptors

A rhodamine B-labelled arginine-rich peptide (RNRHTHLRTRPRK-rhodamine B) was designed as the heparin bioreceptor to construct a highly sensitive and selective fluorescent biosensor for heparin detection. The heparin at picomolar concentration can greatly quenching the fluorescence of the rhodamine B-labelled peptide (RBP) probe in phosphate-buffered solution at pH 5.0. The fluorescence quenching mechanism of the RBP probe by heparin was studied by various analytical techniques like resonance light scattering, fluorescence lifetime, circular dichroism, and UV-vis absorption spectroscopy. The results show that upon binding to heparin, the RBP containing multiple heparin-binding sites undergoes a conformational change from an unfolded form to a folded form that enables two rhodamine B fluorophores come close to each other, leading to the aggregation-caused quenching effect between the RBP and heparin. The developed heparin biosensor displays two linear ranges of 0.01-0.1 nM and 1.0-70.0 nM. The detection limit for heparin detection is measured to be 7.5 pM. In addition, the biosensor exhibits a high selectivity towards heparin over other potential interfering substances. The high sensitivity and good selectivity of the heparin biosensor are very likely attributed to the strong electrostatic interactions of highly negative-charged heparin with the RBP and the spatial conformation of the heparin. The validity of the constructed biosensor for heparin detection is then attested in human serum samples.