Dual Role of a Fluorescent Peptidyl Probe Based on Self-Assembly for the Detection of Heparin and for the Inhibition of the Heparin-Digestive Enzyme Reaction.

The detection of fluorescent probes for biomolecules and control of the function of a complex through a recognition process have not been investigated intensively. A fluorescent peptidyl probe (1) based on the self-assembly stimulated by heparin was synthesized. The fluorescent probe with an aggregation-induced emission fluorophore formed a self-assembling complex with heparin, resulting in a sensitive and selective turn-on response to heparin compared to its biological competitors. The detection limits for heparin were measured to be 138.0 pM (R2 = 0.976) in aqueous solution and 2.6 nM (R2 = 0.996) in aqueous solution containing human serum. Nanosized aggregates formed through the self-assembly of the complex showed potent resistance against the heparin-digestive enzyme. The dual role of the probe for the detection of heparin and the inhibition of heparinase-mediated digestion through the recognition process was used for the real-time monitoring of the enzyme activity of heparinase for the digestion of heparin. Furthermore, the dual role of the probe was applied for the detection of the oversulfated chondroitin sulfate contaminant in heparin.

Pyrene Excimer-Based Peptidyl Chemosensors for the Sensitive Detection of Low Levels of Heparin in 100% Aqueous Solutions and Serum Samples.

Fluorescent chemosensors (1 and 2, Py-(Arg)nGlyGlyGly(Arg)nLys(Py)-NH2, n = 2 and 3) bearing two pyrene (Py) labeled heparin-binding peptides were synthesized for the sensitive ratiometric detection of heparin. The peptidyl chemosensors (1 and 2) sensitively detected nanomolar concentrations of heparin in aqueous solutions and in serum samples via a ratiometric response. In 100% aqueous solutions at pH 7.4, both chemosensors exhibited significant excimer emission at 486 nm as well as weak monomer emission in the absence of heparin. Upon the addition of heparin into the solution, excimer emission increased with a blue shift (10 nm) and monomer emission at 376 nm decreased. The chemosensors showed a similar sensitive ratiometric response to heparin independent of the concentration of the chemosensors. The peptidyl chemosensors were applied to the ratiometric detection of heparin over a wide range of pH (1.5-11.5) using the excimer/momomer emission changes. In the presence of serum, 1 and 2 displayed significant monomer emission at 376 nm with relatively weak excimer emission and the addition of heparin induced a significant increase in excimer emission at 480 nm and a concomitant decrease in monomer emission. The enhanced ratiometric response to heparin in the serum sample was due to the interactions between the peptidyl chemosensors and serum albumin in the serum sample. The detection limits of 2 for heparin were less than 1 nM in 100% aqueous solutions and serum samples. The peptidyl chemosensors bearing two heparin-binding sites are a suitable tool for the sensitive ratiometric detection of nanomolar concentrations of heparin in 100% aqueous solutions and serum samples.