H₂O₂ And Phosphorylated Peptide Dual-response Nanochannel Device

Reactive oxygen species（ROS）are important molecules in many cellular processes,including cell signaling,growth,differentiation and survival.Excessive oxidation will cause cell phosphorylation,leading to many diseases,among which hydrogen peroxide（H₂O₂）,superoxide anion(O₂^·-)and hydroxyl radical（·OH）are the most important oxidizing substances in cells,and the reducing system of antioxidant includes reducing enzymes（such as peroxidase,etc.）,small molecules reducing substances（such as glutathione）,etc.In this paper,we designed a bionic ion nanochannel device that can be accurately identified by GGGCEG（GPGGA）₄CEGRRRR elastic peptide chain to distinguish the responses of different phosphorylated peptides.The nanochannel device consists of a chemically etched polyethylene terephthalate（PET）film with a conical nanochannel and a GGGCEG（GPGGA）₄CEGRRRR functional unit.The peptides were linked to the inner wall of the nanochannel by self-assembly,which could be verified by water contact Angle and X-ray electron spectroscopy.RRRR,the core binding unit in GGGCEG（GPGGA）4CEGRRRR,can form multiple electrostatic interactions with phosphorylpeptides,which trigger the expansion of the polypeptide chain GGGCEG（GPGGA）4CEGRRRR and block transmembrane ion transport,resulting in a significant reduction in ion current in the nanochannel device.By using different ionic current changes,the device can distinguish tyrosine phosphorylpeptide,serine phosphorylpeptide and threonine phosphorylpeptide,and their ionic current changes at-2V are about 56%,40%and 38%,respectively.And to distinguish between single-site and two-site phosphorylpeptides.In addition,the guanidine-phosphate interaction between GGGCEG（GPGGA）₄CEGRRRR and phosphorylated peptides was clearly demonstrated by laser scanning confocal microscopy,adsorption kinetics,electrochemical impedance spectroscopy and molecular interaction analyzer.The oxidation of Cys-XXX-Cys by H2O2results in conformational changes of the polypeptide chain,which can also be verified by108%ion current change rate and circular dichroism.Importantly,the nanochannel enables real-time monitoring of kinase catalyzed phosphorylation and monitoring of reactive oxygen species released by living cells.This work constructs a selective phosphorylated peptide and H₂O₂dual-responsive nanochannel system.