Oxidoreductase Protein Immobilization Technology For Bio-sensors

It's an exciting challenge in the research of biosensors that how bio-macromolecules could be efficiently immobilized on kinds of sensing surfaces with their active conformations during a relatively long period,while facilitating the collection of biochemical reaction signals by transducers promptly and adequately. Redox enzymes catalyze oxidation-reduction reactions,during which measurable signals are easy to be obtained,for example,the transferred electrons are able to be detected by electrochemical instruments,or the color changes are able to be detected by UV-visible spectrum.As a result,redox enzymes are suitable to serve as sensitive biological elements for biosensors,also as model molecules for quantitative evaluation to the effect of enzyme immobilization.Choline oxidase(ChOx),glucose oxidase(GOx),and horseradish peroxidase (HRP),all of which are commonly used redox enzymes to fabricate biosensors,have been selected for the study on two types of strategies of protein immobilization. ChOx and GOx have been immobilized on metal surfaces by self-assembled films of hydrophobins,while HRP has been immobilized in microcapsules based on Layer-by-Layer polyelectrolyte deposition.Hydrophobins are small proteins of about 100 amino acid residues,secreted exclusively by filamentous fungi.There is an approximate 4 nm² planar hydrophobic patch on the surface of hydrophobin molecular,and the rest parts are hydrophilic.The characteristic amphiphilic structure contributes to the extremely high surface activity of hydrophobins,which significantly reduce the surface tension of water and self-assemble into strong,highly ordered films at almost any interfaces,helping fungi attach to various surfaces under different environmental conditions or grow into the air from aqueous environment.On the basis of differences in hydropathy patterns of the amino acid sequences and biophysical properties,two different types of hydrophobins are distinguished,namely classâ… and classâ…¡.The self assembly behavior of a classâ…¡hydrophobin,HFBI from Trichoderma reesei has been investigated in this dissertation.Furthermore,the possibility of HFBI to serve as enzyme immobilization matrix for amperometric biosensors has been studied,in order to construct "all-protein modified" enzyme electrodes and to bring all the catalytic activities of immobilized enzymes into play.Experimental results of quartz crystal microbalance confirmed the self-assembly of HFBI on platinum substrate,indicating that the mass of HFBI self-assembly was barely affected by pH while it increased non-linearly together with the increase of HFBI concentration.The permeability of HFBI films self-assembled on platinum electrode was checked by cyclic voltammetry,whose results were in accordance with those of quartz crystal microbalance.It's believed that solutions of higher HFBI concentrations were apt to form more compact films on platinum surface and exhibited larger self-assembled HFBI mass and stronger block to electrochemical interferences.The optimal HFBI concentration for fabricating biosensors was 20μg/mL.Hydrophilic microenvironments on the biosensing surfaces would help enzymes keep their bioactive conformations.Water contact angle was measured to evaluate the wettability of gold surfaces before and after HFBI modification.The surface hydrophilicity was remarkably and steadily improved after HFBI processing.ChOx was successfully immobilized on HFBI modified gold surface via physical adsorption, with a coverage density of 3366 ng·cm^-2.After immobilization,the optimal pH of ChOx slightly changed from pH 8.0 to pH 7.6,while the apparent Michaelis constant Km^app was 1.27 mM and close to free ChOx.An amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode,which showed low limits of detection of 0.01mM choline(signal-to-noise ratio=3),wide linear range from 0.01 to 1.0 mM,high sensitivity of 2184.06458 nA·mM^-1,also well selectivity and lifetime.Comparing with our choline biosensors previously reported,the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, which produced as large as 4718 nA response current by 0.238μg immobilized ChOx, when saturated by choline substrate.GOx was successfully immobilized on HFBI modified platinum surface via electrostatic adsorption,with a coverage density of 859 ng·cm^-2.The apparent Michaelis constant Km^app of immobilized GOx was 14.8 mM and a little lower than the Km of free ChOx,indicating higher affinity for glucose after immobilization.An amperometric glucose biosensor was constructed based on the Pt/HFBI/GOx electrode,which showed low limits of detection of 0.12 mM glucose(signal-to-noise ratio=3),wide linear range from 0.5 to 20 mM,high sensitivity of 0.29806μA·mM^-1,also well selectivity and lifetime.Comparing with other glucose biosensors previously reported,the HFBI self-assembled film exhibited especially high efficiency of enzyme utilization,which produced at most 710μA responsive current for per unit activity of GOx,when saturated by glucose substrate.The dissertation applied a classâ…¡hydrophobin in amperometric biosensors for the first time.Both of the choline biosensor and the glucose biosensor were of high performances,which thus provided two simple and effective strategies for enzyme immobilization based on hydrophobin HFBI self-assembly.This dissertation also reported a preparation technique of polyelectrolyte microcapsules for steady immobilization of enzymes,with simplified procedures and mild reaction conditions.Negatively charged CaCO₃ microparticles containing HRP molecules were prepared and subsequently enwrapped with layer-by-layer deposited multilayer films,which were constructed via an alternate electrostatic adsorption of poly(allylamine hydrochloride)(PAH) and poly(styrene sulfonate)(PSS).The CaCO₃ templates were then dissolved,leaving homogeneous and well dispersed HRP-(PAH/PSS)₅ microcapsules with an average diameter of about 6μm.During 50 days,the leakage of HRP out of the microcapsules was no more than 6%of the total immobilized HRP amount.While detected by UV-visible spectrum,the encapsulated HRP showed catalytic activity to pyrogallol substrate,the decrease of which was no more than 20%during 30 days.The selective permeability might help with the substrate specificity and the anti-interference capability of the prepared microcapsules. Thus,HRP-containing microcapsules might be promising in biosensor development for detection and treatment of phenolic compounds in environmental pollution.