Protein Macromolecularly Imprinted Alginate Polymeric Microspheres And Their Induce-fit Rebinding Behavior

The protein macromolecularly imprinted polymeric microspheres have become more and more attractive in many fields. The rebinding within macromolecularly imprinted gel polymer systems involves conformational changing and fitting that are different from classical molecularly imprinted materials. This makes it resemble some of the steps in recognition of life system but also possess essential differences.The operations of protein imprinting should take into account the impact of the protein properties, i.e. the volume effect, multi-functional groups and conformational instability and so on. It has become a consensus of the researchers that hydrogels with high water content, suitable mesh scale, numerous functional groups and satisfying swelling properties be used as a new kind of imprinted materials. Calculation and theoretical models applicable for protein imprinting materials are Langmuir monolayer adsorption model, Scatchard analysis, as well as the principle of target interaction mode and imprinting induced fitting model proposed by our group.In this study protein imprinted calcium alginate microspheres were prepared by two novel aqueous suspending methods. The experimental conditions affecting microspheres'morphology, shape distribution and mechanical properties were studied. Optimum preparation program have been made giving consideration to both sides of yield and uniformity. Then protein aggregations and their influencing factors were studies and the required reagent quantity and solution conditions were also investigated.The imprinting matrix was made of gel polymer with different crosslinking density. The imprinting induced fitting behavior was studied by investigating rebinding speed variation as the function of crosslinking density and swelling degree. The imprinting induce-fit model was established accordingly and parameter"fitting factor"was introduced to measure the fitting behavior of the matrix. The existence and predictability of fitting behavior of hydrogel imprinting systems were confirmed by theoretical calculations.Researches in view of the protein template have been conducted from the following aspects. The rebinding in protein solutions with different aggregating states was studied by Scatchard analysis. The dimer related rebinding was studied by means of first order consecutive reaction formula and the regression equation of rebinding quantity was established. The rebinding quantity and rebinding free energy formula of single molecule, dimer, and mixed system were developed from Arrhenius equation. Then an important conclusion was proposed:"the imprinting induced fitting model's essence is the entropic/enthalpic compensation mechanism".The ultimate objective of imprinted polymer materials is specific rebinding. In this study the lysozyme and cytochrome C were templated in hydrogel microspheres with the aim of studying rebinding selectivity. The"relative imprinting efficiency (IEr)"had been proposed to estimate rebinding specificity of imprinted beads in single and double component solutions. As characterized by UV spectrophotometer, conductivity meter and ionic exchange chromatograph, the IEr of mono-imprinted microspheres in double component solutions may reach as high as 2.92 and 3.91, i.e. the selective rebinding is satisfying.