A Protease-Responsive Polymer/Peptide Conjugate and Reversible Assembly of Silver Clusters for the Detection of Porphyromonas gingivalis Enzymatic Activity

Wereport the reversible aggregation of silver nanoparticle (AgNP)assemblies using the combination of a cationic arginine-based peptideand sulfur-capped polyethylene glycol (PEG). The formation and dissociationof the aggregates were studied by optical methods and electron microscopy.The dissociation of silver clusters depends on the peptide sequenceand PEG size. A molecular weight of 1 kDa for PEG was optimal forthe dissociation. The most important feature of this dissociationmethod is that it can operate in complex biofluids such as plasma,saliva, bile, urine, cell media, or even seawater without a significantdecrease in performance. Moreover, the peptide-particle assembliesare highly stable and do not degrade (or express of loss of signalupon dissociation) when dried and resolubilized, frozen and thawed,or left in daylight for a month. Importantly, the dissociation capacityof PEG can be reduced via the conjugation of a peptide-cleavablesubstrate. The dissociation capacity is restored in the presence ofan enzyme. Based on these findings, we designed a PEG-peptide hybridmolecule specific to the Porphyromonas gingivalis protease RgpB. Our motivation was that this bacterium is a key pathogenin periodontitis, and RgpB activity has been correlated with chronicdiseases including Alzheimer's disease. The RgpB limit of detectionwas 100 pM RgpB in vitro. This system was used tomeasure RgpB in gingival crevicular fluid (GCF) samples with a detectionrate of 40% with 0% false negatives versus PCR for P. gingivalis (n = 37). The combination of PEG-peptide and nanoparticlesdissociation method allows the development of convenient proteasesensing that can operate independently of the media composition.