H₂O₂ and Phosphorylated Peptide Dual-Responsive Nanochannel Device

Oxidation and protein phosphorylation are critical mechanismsinvolvedin regulating various cellular activities. Increasing research hassuggested that oxidative stress could affect the activities of specifickinases or phosphatases, leading to alterations in the phosphorylationstatus of certain proteins. Ultimately, these alterations can affectcellular signaling pathways and gene expression patterns. However,the relationship between oxidation and protein phosphorylation remainscomplex and not yet fully understood. Therefore, the development ofeffective sensors capable of detecting both oxidation and proteinphosphorylation simultaneously presents an ongoing challenge. To addressthis need, we introduce a proof-of-concept nanochannel device thatis dual-responsive to both H2O2 and phosphorylatedpeptide (PP). Specifically, we design a peptide GGGCEG(GPGGA)(4)CEGRRRR, which contains an H2O2-sensitiveunit CEG, an elastic peptide fragment (GPGGA)(4), and a phosphorylationsite recognition fragment RRRR. When the peptides are immobilizedon the inner walls of conical nanochannels in a polyethylene terephthalatemembrane, this peptide-modified nanochannel device exhibits a sensitiveresponse to both H2O2 and PPs. The peptide chainsundergo a random coil-to-& alpha;-helix transition in response to H2O2, which leads to a close-to-open transition ofthe nanochannel, accompanied with a remarkable increase in the transmembraneionic current. In contrast, binding of the peptides with PPs shieldsthe positive charge of the RRRR fragments, causing a decrease of thetransmembrane ionic current. These unique features enable the sensitivedetection of reactive oxygen species released by 3T3-L1 cells stimulatedby platelet-derived growth factor (PDGF) as well as PDGF-induced changein the PP level. Real-time kinase activity monitoring further confirmsthe device's potential utility for kinase inhibitor screening.