title: Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method.
abstract: Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities. The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers. With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03+/-0.89 x 10(-11)mole/cm(2) of template from a 5.68 x 10(-7)M myoglobin solution, compared to 2.58+/-0.02 x 10(-11)mole/cm(2) for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 degrees C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal. The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K(d)) for the specific binding phase to be 3.4 x 10(-7)M and the binding site capacity to be 7.24 x 10(-11)mole/cm(2). For the non-specific binding phase, K(d) was found to be 1.355 x 10(-5)M and the binding site capacity was determined as 9.62 x 10(-10)mole/cm(2). Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 x 10(-7)M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.
authors.affiliation: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan, ROC.
authors.forename: Hsin-Yi
authors.initials: HY
authors.lastname: Lin
authors.forename: John
authors.initials: J
authors.lastname: Rick
authors.forename: Tse-Chuan
authors.initials: TC
authors.lastname: Chou
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D020366:Methylmethacrylate / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011109:Polymethacrylic Acids / Q000737:chemistry*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D003432:Cross-Linking Reagents; D009211:Myoglobin; D011109:Polymethacrylic Acids; C020946:triethylene glycol dimethacrylate; D020366:Methylmethacrylate; D011092:Polyethylene Glycols