Orientated Immobilization Of Proteins: Bio-affinity And LB Method

Grafted biomaterials, biosensors and bio-batteries have attracted extensiveattention, as advanced technology. These products have been applied in medicalmaterials, environmental protection, food and other fields. The researching andapplications of these products both involved in the interactions of protein and supportmaterials. Without controlled, the distribution of adsorbed protein will random anddisordered. And due to the interaction with the support, adsorbed protein alwayschange its conformation and loss its activity easily. At present, it is still an importantaspect to control the adsorption of protein, including the orientation, amount andconformation of adsorbed protein, to fully express the bio-function of adsorbedprotein.The methods of controlling protein adsorption mainly include physical adsorption,chemical fixation and bio-affinity. But it is hard to control the amount andconformation of adsorbed protein by general physical adsorption method, and toselect the kind of adsorbed protein. The operation of chemical fixation is relativelycomplicated, and the conformation of adsorbed protein easily varify and lead to lossits activity because of the interaction of chemical reagents. Bio-affinity is alsocomplexes, but it could tailor the orientation of adsorbed protein, maintain itsbio-activity, and reuse in some cases.In this work, electrochemical sensors were prepared through bio-affinity method.Firstly, to fabricate the modified cofactors of enzyme, the cofactor (hemin) ofhorseradish peroxidase (HRP) and the cofactor (flavin adenine dinucleotide, FAD) ofglucose oxidase (GOx) were respectively fixed on the functioned grapheme oxide.Then the recombined enzymes were obtained by the recombination of the modifiedcofactor and apo-enzyme. Enzyme electrodes were prepared and the electrochemicalactivities were detected. It could be found that the electrochemical activity ofmodified HRP increased nearly4times than that of the unmodified HRP, whichmodified through graphene oxide and pent-polyaniline. With the reduction of the modified cofactor, the currents increased with the degree of reduction, and the maxcurrent was280μA. And the electrochemical activity of modified GOx increasednearly3times than unmodified, which modified through graphene oxide. With thereduction of the modified cofactor, the currents increased with the degree ofreduction, and the max current was220μA.Tyrosinase was orientated immobilized on the surface of supports byLangmuir-Blodgett (LB) method. Firstly, the surfactant competitive inhibitor oftyrosinase was synthesized, then this molecule combined with tyrosine and preparedLB films, the catalytic activities were measured. Then the water-soluble polyanilinewas added to fabricate enzyme electrode. It could be found that the activity ofimmobilized tyrosinase by LB method was higher than that by common adsorbedmethod. Furthermore, the activity of tyrosine LB films prepared through mixedcompetitive inhibitor was higher than that without competitive inhibitor. Afteradding polyaniline, the electrochemical activity was increased and the oxidationvoltage and overpotential were reducing, the catalytic current and sensitivity oftyrosinase electrode were improved.A series of fibrin (ogen) and bovine serum albumin binary protein LB films wereprepared. Then the effect of different ratios proteins on the behavior of platelet wasinvestigated. It could be found that HFg changed into HF through general adsorptionmethod. And the conformation of HFg could be maintained by LB method. A seriesof HFg/BSA films were obtained by general method, which could cause plateletadhesion and deformation, and formed network structure. HFg/BSA films fabricatedby LB method would not induce platelet adhesion and deformation. HF/BSA filmsprepared by LB method would not induce platelet adhesion and all platelet keptspherical, when the proportion of HF was25%; as the proportion of HF increased to49%, platelet adhered, extended and formed pseudopods. The reasons for this may bethe C chains of HF have the interaction points which interact with integrin ofplatelet, or the C chains formed network structure and HF could interact withplatelet under the structure.