Preparation Of Biomimetic Ion Channel Based On Polymer And Research Of Its Response To CAMP

As a membrane protein,the biological ion nanochannel widely exists in the biological system.It can generate conformational changes in response to external stimulation to regulate the ion transport on the cell membrane with high selectivity,and then play an extremely important role in the process of signal transduction,energy conversion,potential regulation,material exchange and function regulation.Inspired by biological nanochannels,a series of intelligent and functional artificial nanochannels have been explored to simulate biological processes in vitro.Similar to biological nanochannels,these artificial nanochannels as a promising platform,have been widely used in gas detection,drug delivery,nucleic acid analysis,ion permeation,etc.Therefore,it is very important to develop a new type of artificial ion nanochannel based on smart polymer for the realization of biological functions by making full use of the stability,designability and expansibility of the artificial nanochannels and the responsiveness and functionality of smart polymer.In this paper,inspired by the biological ionchannel,we take advantage of the conformational change of polymer to simulate the conformational change of ion channel protein in the process of ion transport.From the molecular level to the polymer level and then to the construction of nanochannels,step by step,a novel cAMP–regulated artificial nanochannel based on a tripeptide Arg-Thr-Ala（abbreviated as RTA）design is developed.The details are as follows:1.Firstly,the tripeptide RTA responding to cAMP were screened out by searching the Protein Data Bank（PDB）.Then the binding mode of the hydrogen bond interaction between RTA and cAMP was preliminarily verified from the molecular level through the crystal structure analysis,nuclear magnetic titration experiment and the two-dimensional nuclear magnetic experiment,and the strong binding ability between RTA and cAMP was verified by the fluorescence titration experiment.The binding constant（K_a）was 3751 L·mol^?1,which was much higher than that of ATP(32L·mol^?1),ADP(11 L·mol^?1),AMP(450 L·mol^?1)and adenosine(54 L·mol^?1).2.According to the design concept of recognition unit-function conversion unit,a two component copolymer PNI-co-ARTA_0.1 with tripeptide RTA as recognition unit and poly（N-isopropylacrylamide）（PNIPAAm）as function conversion unit was designed and prepared.PNIPAAm provides a flexible polymer backbone,which promotes the conformational transition of the copolymers in response to cAMP binding.The conformational change of the copolymers were further characterized by the measurements of lower critical solution temperature（LCST）and isothermal titration calorimetry（ITC）.The selective adsorption behavior of copolymers to cAMP and the change of electrochemical properties of polymer film surface were characterized by grafting the copolymer onto the surface of QCM sensors and Au electrode in turn for Quartz crystal microbalance（QCM-D）test and electrochemical impedance spectroscopy（EIS）measurements.3.Finally,PNI-co-ARTA_0.1 was grafted into the channels of anodic aluminum oxide（AAO）membrane and Polyethylene terephthalate（PET）membrane by surface initiated atom transfer radical polymerization（SI-ATRP）and surface chemical grafting,respectively,to construct the symmetrical and asymmetric modified nanochannels in response to cAMP.The surface morphology was characterized by atomic force microscopy（AFM）and scanning helium ion microscopy（HIM）,and their ion transport properties were characterized by picoammeter.All results show that the nanochannel can not only detect the ultra-low concentration(pmol?L^?1)of cAMP,but also has good selectivity and excellent reversibility.It will open up a new way to realize the accurate recognition of cAMP and has a better understanding of the binding behavior of cAMP in biological ion channels.